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.     

Dual-plane ultrafast limited-angle electron beam x-ray tomography

Electron beam x-ray tomography is an imaging technique, which can provide cross-sectional images of an object of interest with about 1 mm spatial resolution at frame rates of up to 10,000 frames per second. As a non-intrusive method it is especially suited for studying multiphase flows. For this purpose we devised an experimental limited-angle scan setup which utilizes linear beam deflection to generate radiographic projections. This setup was employed in the study of gas–liquid flow in an experimental flow loop operated at different liquid and gas flow rates. Electron-beam tomography images were compared with image data of a wire-mesh sensor. The latter is a fast but intrusive imaging device which is commonly used in gas–liquid flow imaging and achieves comparable frame rates but at lower spatial resolution. As a novelty we implemented a dual-plane limited-angle electron beam x-ray tomography which allows us to gain information about the phase velocities using cross-correlation data analysis.     

Atomic force microscopy at ambient and liquid conditions with stiff sensors and small amplitudes

We report on atomic force microscopy (AFM) in ambient and liquid environments with the qPlus sensor, a force sensor based on a quartz tuning fork with an all-electrical deflection measurement scheme. Small amplitudes, stiff sensors with bulk diamond tips and high Q values in air and liquid allow to obtain high resolution images. The noise sources in air and liquid are analyzed and compared for standard silicon cantilevers and qPlus sensors. First, epitaxial graphene was imaged in air, showing atomic steps with 3 A? height and ridges. As a second sample system, measurements on calcite (CaCO(3)) in liquids were performed in water and polyethylenglycol (PEG). We demonstrate high resolution images of steps in PEG on calcite and nanolithography processes, in particular with frequency-modulation AFM the controlled dissolution of calcite monolayers.     

Design and use of a computer controlled confocal microscope for biological applications

In the confocal scanning light microscope where the object is scanned mechanically through a finely focused laser spot, a fundamentally higher resolution can be achieved when a point detector is employed. Such a microscope possesses in addition to a high dynamic range excellent sectioning capabilities, and is because of this very suitable for “3-Dimensional imaging”, especially when operated in fluorescence. An instrument under computer control and with extensive image processing capabilities is described. Various biological applications are given including computer generated stereo images of biological structures with submicron resolution.     

A novel atomic force microscope operating in liquid for in situ investigation of electrochemical preparation of porous alumina

A novel atomic force microscope (AFM) operating in liquid is described in this article. The specially designed AFM probe involves a tip attached to a cantilever, a tip holder, and a circular Plexiglas window. When the probe dives into the fluid, a circular meniscus is established around the Plexiglas window, preventing the tip from being affected or destroyed by surface tension of the liquid. In this setup, the whole scanning probe and the sample can completely dive into fluid. Meanwhile, the probe tip scans over the sample surface when the instrument works. These advantages enable the instrument to scan comparatively large or heavy samples with a high speed. The highest scan rate is about 30 lines/s or 14 s for a 400 x 400-pixel, 3 x 3 microm image. Using the new AFM, we carry out in-situ investigation of the formation processes of porous alumina during electrochemical anodic oxidation. A lead ring and an aluminum foil serve as cathode and anode, respectively. They are entirely immersed in the bath electrolyte, which is oxalic acid solution. During anodic oxidation, the AFM images of the sample surface are successively acquired without elevating the sample out of the solution. Experiments reveal that electrochemical reactions take place soon after the power supply is switched on, and with the progression of anodization, nanostructures of porous alumina gradually occur on the aluminum substrate, finally yielding ordered arrays of nanopores. As a typical example of applications, the results of this work show that the new AFM is an ideal and powerful tool for in-situ observation and study of materials or samples in aqueous solutions.     

基于 LSTM 的矩形纳米光栅 AFM 图像复原方法

原子力显微镜(AFM)利用探针与待测物之间的交互作用力进行成像,通过获取矩形纳米光栅计量标准器具的高分辨率成像得到相关的几何量参数并进行标定,实现从标准计量器具到工作计量器具的量值传递。在AFM扫描过程中,由于针尖的影响作用,使得扫描所获图像是探针和样品共同作用的结果,而不是样品形貌的真实描述。针对这一现象,本文提出了一种基于长短期记忆网络(LSTM)的AFM图像复原方法,该方法对通过膨胀法获得的仿真图像各扫描行进行训练,进而获得适用于矩形纳米光栅AFM图像复原模型。实验结果表明,针对线宽20 nm,高40 nm的矩形纳米光栅,经过该方法复原后光栅线宽的相对误差为7.40%,相较于传统的复原方法进一步提高了测量准确度。     

A compact CCD‐monitored atomic force microscope with optical vision and improved performances

A novel CCD‐monitored atomic force microscope (AFM) with optical vision and improved performances has been developed. Compact optical paths are specifically devised for both tip‐sample microscopic monitoring and cantilever's deflection detecting with minimized volume and optimal light‐amplifying ratio. The ingeniously designed AFM probe with such optical paths enables quick and safe tip‐sample approaching, convenient and effective tip‐sample positioning, and high quality image scanning. An image stitching method is also developed to build a wider‐range AFM image under monitoring. Experiments show that this AFM system can offer real‐time optical vision for tip‐sample monitoring with wide visual field and/or high lateral optical resolution by simply switching the objective; meanwhile, it has the elegant performances of nanometer resolution, high stability, and high scan speed. Furthermore, it is capable of conducting wider‐range image measurement while keeping nanometer resolution. Microsc. Res. Tech. 76:931–935, 2013. © 2013 Wiley Periodicals, Inc.     

运用碳纳米管原子力显微镜针尖减小长程力作用的研究

减小探针和样品表面之间的长程宏观力是原子力显微镜获得高分辨率成像的关键。首先通过理论分析得出影响长程力的主要因素是探针的几何形状和尺寸。然后分别运用几何形状和尺寸不同的原子力显微镜的传统Si针尖和碳纳米管针尖对样品进行扫描试验研究,结果显示碳纳米管针尖较传统针尖获得了高分辨率的图像。这一结果表明,碳纳米管针尖减小了成像中宏观长程作用力的影响,是理想的原子力显微镜针尖。     

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330-337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ～8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ～4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).     

A Novel Atomic Force Microscope with High Stability and Scan Speed

Abstract

A novel atomic force microscope (AFM) has been developed. Unlike conventional AFM systems, its cantilever and tip were set up in the X direction with respect to the sample. It can practically eliminate the crawling effect of the probe itself. Meanwhile, using a beam splitter, we devised a unique path optical beam deflection method for the measurement of the cantilever's displacement with its minimized structure and an optimal light‐amplifying ratio; the position of the sensitive detector (PSD) is just in front of the visual sight of operator. This makes observation and operation easier. Furthermore, the PSD attached to the translation stage can be adjusted and, thus, adapts the setpoint of imaging force to different samples. In this way, our new AFM provides high stability and scan speed. The highest scan rate is about 40 lines/s or 10s for a 400×400‐pixel, 3 µm×3 µm image.     

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 imaging using a tip-on-chip: opening the door to integrated near field nanotools

We describe in detail how atomic force microscopy (AFM) images can be routinely achieved with macroscopic silicon-based chips integrating mesoscopic tips, paving the way for the development of new near field devices combining AFM imaging with any kind of functionality integrated on a chip. The chips have been glued at the end of the free prong of 100 kHz quartz tuning forks mounted in Qplus configuration. Numerical simulations by modal analysis have been carried out to clarify the nature of the vibration modes observed in the experimental spectra. It is shown that two low frequency modes can be used to drive the system and scan the surface with a great stability in amplitude modulation as well as in frequency modulation AFM under ultrahigh vacuum. The AFM capabilities are demonstrated through a series of examples including phase and dissipation contrast imaging, force spectroscopy measurements, and investigations of soft samples in weak interaction with the substrate. The lateral resolution with the tips grown by focused ion beam deposition already matches the one achieved in standard amplitude modulation mode AFM experiments.     

基于平板扫描器的新型AFM系统

研制了一种基于平板扫描器的新型原子力显微镜(AFM)系统。该系统创新地把二维平板扫描器和一维反馈控制器相结合,有效地克服了传统扫描器Z向反馈控制与XY扫描平面之间的非线性交叉耦合误差,同时保证了大范围扫描时检测光路的稳定性。利用该系统与传统AFM作了氧化铝薄膜和光栅对比扫描实验,结果表明这种AFM系统能够获得无扭曲、规则的理想图像。     

A new confocal scanning beam laser MACROscope using a telecentric,f-theta laser scan lens

A new confocal scanning beam system (MACROscope) that images very large-area specimens is described. The MACROscope uses a telecentric, f-theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small-area, high-speed, high-resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large-area, high-speed, good-resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected-light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.     

MAC mode atomic force microscopy studies of living samples, ranging from cells to fresh tissue

Magnetic AC mode (MAC mode) atomic force microscopy (AFM), a novel type of tapping mode AFM in which the cantilever is driven directly by a magnetic field, is a powerful tool for imaging with high spatial resolution and better signal-to-noise in liquid environment. It may largely extend the application of AFM to living samples, especially those are sensitive to cantilever forces, even to multilayer tissue samples. However, there are few reports on the imaging of living cells by MAC mode AFM previously. In our present study, we explore the optimal imaging conditions of MAC mode AFM on living astrocytes and fresh arterial intima surface. We also used nude tips for PicoTREC panel (i.e., Aux in BNC, a new data collecting channel) to image living samples and discussed its difference with phase imaging. We show that living biological samples can be imaged by MAC mode AFM at details of comparable resolution as those by high resolution scanning electron microscopy. Furthermore, the combination of height, amplitude, phase and TREC panel signals provide abundant informations for the characteristics of living samples, such as topography, profile, stiffness and adhesion.     

Optimization of specimen preparation of thin cell section for AFM observation

High resolution imaging of intracellular structures of ultrathin cell section samples is critical to the performance of precise manipulation by atomic force microscopy (AFM). Here, we test the effect of multiple factors during section sample preparation on the quality of the AFM image. These factors include the embedding materials, the annealing process of the specimen block, section thickness, and section side. We found that neither the embedding materials nor the temperature and speed of the annealing process has any effect on AFM image resolution. However, the section thickness and section side significantly affect the surface topography and AFM image resolution. By systematically testing the image quality of both sides of cell sections over a wide range of thickness (40-1000 nm), we found that the best resolution was obtained with upper-side sections approximately 50-100 nm thick. With these samples, we could observe precise structure details of the cell, including its membrane, nucleoli, and other organelles. Similar results were obtained for other cell types, including Tca8113, C6, and ECV-304. In brief, by optimizing the condition of ultrathin cell section preparation, we were able to obtain high resolution intracellular AFM images, which provide an essential basis for further AFM manipulation.     

Differential contrast imaging of secondary electron signals in cryo-field-emission scanning electron microscopy

Low-temperature scanning electron microscopy (LTSEM) is limited in resolution and image quality by charging of frozen hydrated samples and collection deficiencies of secondary electron signal contrasts. We measured and corrected both effects using differential hysteresis processing (DHP) of LTSEM images, scanned at 15-bit from 5×4 inch Polaroid negatives. Bulk charging produced a major contrast component equal to 44–87% of the intensity range of the image. The strong charging contrast reduced the local high-resolution signal contrasts to an unrecognizable level. Segmentation and imaging of the unaffected surface contrasts produced high-quality images of high contrast from metal-coated samples as well as from uncoated samples. The differential contrast imaging can be used for control of the sequential etching of ice from the non metal-coated sample as well as improved LTSEM imaging of the finally coated sample.     

Dynamic force microscopy imaging of native membranes

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.     

Imaging with neutral atoms—a new matter-wave microscope

Matter‐wave microscopy can be dated back to 1932 when Max Knoll and Ernst Ruska published the first image obtained with a beam of focussed electrons. In this paper a new step in the development of matter‐wave microscopy is presented. We have created an instrument where a focussed beam of neutral, ground‐state atoms (helium) is used to image a sample. We present the first 2D images obtained using this new technique. The imaged sample is a free‐standing hexagonal copper grating (with a period of about 36 μm and rod thickness of about 8 μm). The images were obtained in transmission mode by scanning the focussed beam, which had a minimum spot size of about 2.0 μm in diameter (full width at half maximum) across the sample. The smallest focus achieved was 1.9 ± 0.1 μm. The resolution for this experiment was limited by the speed ratio of the atomic beam through the chromatic aberrations of the zone plate that was used to focus. Ultimately the theoretical resolution limit is set by the wavelength of the probing particle. In praxis, the resolution is limited by the source and the focussing optics.     

Atomic force microscope tip cleaning by using gratings as micro‐washboards

The sharpness of atomic force microscope (AFM) tips is essential for acquiring high quality AFM images. However, AFM tips would easily get contaminated during scanning and storage at ambient condition, which influences image resolution and causes image distortion. Replacing the probe frequently is a solution, but uneconomical. To solve this problem, several tip cleaning methods have been proposed but there is space for further improvement. Therefore, this article developed a method of tip cleaning by using a one‐dimensional grating (600 lines/mm) as a micro‐washboard to “wash” contaminated tips. We demonstrate that the contaminants can be scrubbed away by rapidly scanning such micro‐washboard against the tip in the aids of Z‐dithering (10–20 Hz) exerted on the washboard. This method is highly efficient and proved to be superior to traditional ones. Experiments show that AFM images acquired with “washed” tips have higher resolution and less distortion compared with images acquired using contaminated tips, even comparable to those scanned by new ones. Microsc. Res. Tech. 76:1131–1134, 2013. © 2013 Wiley Periodicals, Inc.