Position referencing in optical microscopy thanks to sample holders with out-of-focus encoded patterns

This article introduces smart sample holders for optical microscopy. Their purpose is to allow the absolute determination of the position of the observed zone with respect to the sample holder itself and with a high accuracy. It becomes then straightforward to find a given zone of interest by positioning coarsely the microscope slide to the same position coordinates. Furthermore images recorded during different observation sessions; i.e. for slightly different positions; can be processed numerically in order to superimpose them with a high accuracy. Thus the slight deviations of the microscope slide position and orientation due to the different observations are compensated numerically and a perfect superimposition of the recorded images is performed. Then accurate site-by-site image comparisons become possible even for images recorded during different observation sessions and over a long period of time. The subpixel capability of the proposed method is demonstrated and those smart microscope slides constitute a new tool for live cell experiment.
In practise, an encoded geometrical pattern used as position reference is inserted in a plane parallel to the surface receiving the tissue section or sample. Then the transition of the focus position from the tissue section to the position reference requires only a vertical adjustment and does not affect the lateral coordinates of observation. The numeric processing of the image of the position reference pattern allows the retrieval of the lateral coordinates that are also used for the tissue section image. Thus each image is recorded with a set of position coordinates that defines accurately the position of the observed area with respect to the sample holder itself.     

Atomic force microscopy of purple membranes

The surface structure of purple membranes was imaged using an atomic force probe mounted in a scanning tunnelling microscope. One of the two different membrane surfaces showed protruding, disc-shaped features forming an hexagonal lattice with about 6 nm centre to centre spacing. These are identified as the cytoplasmic surfaces of trimers of bacteriorhodopsin molecules and are correlated with the structural information on bacteriorhodopsin obtained from numerous earlier electron microscope and diffraction studies.     

Design of a scanning probe microscope with advanced sample treatment capabilities: An atomic force microscope combined with a miniaturized inductively coupled plasma source

We describe the design and performance of an atomic force microscope (AFM) combined with a miniaturized inductively coupled plasma source working at a radio frequency of 27.12 MHz. State-of-the-art scanning probe microscopes (SPMs) have limited in situ sample treatment capabilities. Aggressive treatments such as plasma etching or harsh treatments such as etching in aggressive liquids typically require the removal of the sample from the microscope. Consequently, time consuming procedures are required if the same sample spot has to be imaged after successive processing steps. We have developed a first prototype of a SPM which features a quasi in situ sample treatment using a modified commercial atomic force microscope. A sample holder is positioned in a special reactor chamber; the AFM tip can be retracted by several millimeters so that the chamber can be closed for a treatment procedure. Most importantly, after the treatment, the tip is moved back to the sample with a lateral drift per process step in the 20 nm regime. The performance of the prototype is characterized by consecutive plasma etching of a nanostructured polymer film.     

用于外场试验的交会测量

提出结合坐标系转换和按行独立的加权总体最小二乘法(RWTLS)的交会测量方法用于外场试验。该方法利用空间坐标系转换方法获得目标点在大地坐标下的空间角度参数;通过多余观测数建立条件方程确定起算数据间的角度位置关系,用RWTLS和高斯-牛顿迭代方法求得运动目标点在任一时刻的空间角度坐标;最后,利用对静态目标点的观测获得基距,结合目标点的空间角度坐标求得其轨迹曲线。实验结果表明:观测站的坐标误差在±0.15m以内,运动目标在X、Y、Z方向上的坐标误差在±0.4m内。与传统的两站前方交会测量方法相比,该方法无须校正经纬仪坐标系,也不必已知观测站位置参数,从而减少了对起算数据的需求,减少了布站工作量,具有直接简便、收敛速度快、精度较高等优点,在飞行目标外场测试中有良好的实用性。     

Preparation of frozen-hydrated specimens for high resolution electron microscopy

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.     

Diagnosis of aberrations from crystalline samples in scanning transmission electron microscopy

The Ronchigrams, or shadow images, observed from a thin crystalline sample in a scanning transmission electron microscope characteristically present many sets of fringes, which appear thanks to the coherent interference between the various Bragg-diffracted discs as they overlap in the diffraction plane. A particular region of such patterns can be shown to be independent of the defocus at which they are recorded. The intensity along this so-called achromatic ring depends on the microscope aberrations and can be used to diagnose the wave aberration coefficients, a crucial first step in the operation of an aberration-corrected microscope. A new algorithm is presented that allows the accurate determination of all non-cylindrically symmetric aberrations up to fourth-order from a crystalline sample using this property of the Ronchigram. An experimental procedure for determining the position of and intensity along the achromatic lines, as well as examples of diagnosis from two different crystalline structures, are detailed.     

Diagnosis of aberrations from crystalline samples in scanning transmission electron microscopy

The Ronchigrams, or shadow images, observed from a thin crystalline sample in a scanning transmission electron microscope characteristically present many sets of fringes, which appear thanks to the coherent interference between the various Bragg-diffracted discs as they overlap in the diffraction plane. A particular region of such patterns can be shown to be independent of the defocus at which they are recorded. The intensity along this so-called achromatic ring depends on the microscope aberrations and can be used to diagnose the wave aberration coefficients, a crucial first step in the operation of an aberration-corrected microscope. A new algorithm is presented that allows the accurate determination of all non-cylindrically symmetric aberrations up to fourth-order from a crystalline sample using this property of the Ronchigram. An experimental procedure for determining the position of and intensity along the achromatic lines, as well as examples of diagnosis from two different crystalline structures, are detailed.     

Application of the focused ion beam technique in aerosol science: detailed investigation of selected, airborne particles

The focused ion beam technique was used to fabricate transmission electron microscope lamellas of selected, micrometre‐sized airborne particles. Particles were sampled from ambient air on Nuclepore polycarbonate filters and analysed with an environmental scanning electron microscope. A large number of particles between 0.6 and 10 µm in diameter (projected optical equivalent diameter) were detected and analysed using computer‐controlled scanning electron microscopy. From the resulting dataset, where the chemistry, morphology and position of each individual particle are stored, two particles were selected for a more detailed investigation. For that purpose, the particle‐loaded filter was transferred from the environmental scanning electron microscope to the focused ion beam, where lamellas of the selected particles were fabricated. The definition of a custom coordinate system enabled the relocation of the particles after the transfer. The lamellas were finally analysed with an analytical transmission electron microscope. Internal structure and elemental distribution maps of the interior of the particles provided additional information about the particles, which helped to assign the particles to their sources. The combination of computer‐controlled scanning electron microscopy, focused ion beam and transmission electron microscopy offers new possibilities for characterizing airborne particles in great detail, eventually enabling a detailed source apportionment of specific particles. The particle of interest can be selected from a large dataset (e.g. based on chemistry and/or morphology) and then investigated in more detail in the transmission electron microscope.     

Alignment of AFM images using an iterative mathematical procedure

An iterative mathematical procedure for the alignment of sequentially recorded atomic force microscope images (AFM) is presented. The computer program is able to correct commonly observed drifts in vertical and lateral directions, rotations around a vertical or lateral axis and differences in scale. This method is applied on dissolution experiments of uranium dioxide (UO2) surfaces. Images recorded during in situ experiments, which are shifted probably due to thermal fluctuations, can be aligned with good accurancy. In a further approach the UO2 surface is marked by electron-beam-induced deposition (EBD or EBID) with microstructured reference points. The alignment can be distinctly improved using marked sample surfaces because of the characteristic shape of the markings, which do not change during the experiment. Furthermore, the markings can be used to find again a domain on a sample surface. Therefore, AFM images recorded before and after an ex situ experiment (e.g. treatment in corrosive medium for a longer period of time) can be aligned with a nanometer spatial resolution.     

Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy

We report a simple methodology to provide complete pulse characterization at the sample plane of a two-photon excited fluorescence (TPEF) microscope. This is achieved by using backward propagating second-harmonic generation (SHG) from starch granules. Without any modification to the microscope, SHG-autocorrelation traces were obtained by using a single starch granule that was placed alongside the biological specimen being imaged. A spectrally resolved SHG autocorrelation was acquired by placing a spectrometer at the output port of the microscope. Complete in situ pulse information is then directly retrieved in an analytical way using the measurement of electric filed by interferometric spectral trace observation (MEFISTO) technique.     

一种目标位置解算方法

目标位置解算装置是快速、准确地获取空中或地面目标位置坐标的有效手段,由激光测距机提供目标距离参数,由GPS接收机获得观测点坐标参数,方位获取设备提供目标方位角及高低角参数。在目标位置解算过程中,建立目标局部坐标系,根据地球的椭球模型,建立相应的地心坐标系。根据求得的目标局部坐标系与地心坐标系的转换关系,解算空中或地面目标相对于地心坐标系的大地坐标参数。通过对已知WGS-84坐标系数值的地面固定目标进行测试,在30 km的探测距离上,达到了目标定位误差绝对值小于1％的精度要求。     

Ultramicrotomy in the ESEM, a versatile method for materials and life sciences

We here present the results of the first materials science analyses obtained with the prototype of a serial block-face sectioning and imaging tool, 3View™ of Gatan, Inc (Pleasanton, CA, U.S.A.). It is a specially designed ultramicrotome operating in situ within an environmental scanning electron microscope originally developed for life science research. The microtome removes thin slices from the sample and the environmental scanning electron microscope images each new block surface of the specimen (serial block-face scanning electron microscopy). The Schottky emitter (FEG) of the microscope delivers high spatial resolution and has the advantage of stable performance and high durability. The slice thickness can typically be selected between 50 and 100 nm. It is possible to cut hundreds of slices and simultaneously acquire images with Digital Micrograph™ Model 700 (Gatan, Inc.). This article outlines the set-up and describes the automated process. The preparation of specimens for in situ ultramicrotomy is explained and the parameters for good image quality are discussed. In addition, special operative and analytic features of the controlling software are presented. Three different technical materials and one botanical specimen were analyzed delivering first results of this method for materials science and for botany.     

野外大视场单相机空间坐标测量系统的快速标定

针对靶场现场监测范围大,相机焦距不固定,相机空间位置及角度各不相同的情况,为实现对弹落点空间坐标位置的高精度自动测量,提出了一种在野外大视场环境下使用的基于单相机空间坐标测量系统的快速标定方法。首先,在小孔成像模型的基础上,通过GPS测量获得视场内两个标定点及相机在大地坐标系中的坐标;然后读取标定点的像素坐标,根据对角相等及最小二乘法实现焦距与旋转矩阵的分步标定;最后在保证标定精度前提下,略去主点的标定过程,确定相机主点为理想主点位置。实验结果表明,在测试距离1km以外,对视场宽度为200m的区域进行监测时,校验点相对定位误差低于0.25%。该相机标定方法不需要高精度靶标,操作简单,适用于野外大视场环境下单相机空间坐标测量系统的快速标定。     

A TDOA-Based Ultrasonic Absolute Localizing System of a Rail Robot in Greenhouse

In this paper,we present a method for localization of a rail autonomous pesticide spraying and sampling robot working in greenhouse using an absolute localization system.Design and implementation of the localization system comes from the usage of beacon systems each of which is composed of an RF single receiver and an ultrasonic transmitter.The RF single receiver gets the synchronization signal from the mobile robot,and the ultrasonic transmitter sends ultrasonic signal,thus the distance from the beacon to the ultrasonic receiver can be measured.The position of a beacon in coordinate system of robot can be calculated according to distance information from the beacons to two ultrasonic receivers which are mounted on the robot.Based on the coordinate transformation,the position of a mobile robot can be calculated from the beacon’s absolute position information in the global coordinate system.Experiments demonstrate the effectiveness of the proposed method in real world applications.     

Compact very low temperature scanning tunneling microscope with mechanically driven horizontal linear positioning stage

We describe a scanning tunneling microscope for operation in a dilution refrigerator with a sample stage which can be moved macroscopically in a range up to a cm and with an accuracy down to the tens of nm. The position of the tip over the sample as set at room temperature does not change more than a few micrometers when cooling down. This feature is particularly interesting for work on micrometer sized samples. Nanostructures can be also localized and studied, provided they are repeated over micrometer sized areas. The same stage can be used to approach a hard single crystalline sample to a knife and cleave it, or break it, in situ. In situ positioning is demonstrated with measurements at 0.1 K in nanofabricated samples. Atomic resolution down to 0.1 K and in magnetic fields of 8 T is demonstrated in NbSe(2). No heat dissipation nor an increase in mechanical noise has been observed at 0.1 K when operating the slider.     

High-angle annular dark-field STEM observation of Xe nanocrystals embedded in Al

High-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) observation of Xe precipitates embedded in crystalline membranes has been made using electron probes of atomic dimensions and HAADF-STEM images of Xe precipitates qualitatively different from conventional TEM observation results have been obtained. Multislice-based HAADF-STEM simulation has been made and it has been revealed that the intensity of images of Xe atoms at positions displaced from Al matrix columns decreases rapidly as the thickness increases. Even in a thin specimen, the off-site Xe atoms of the precipitate at deep locations, were not observable. Therefore, different images are expected for specimens of different thicknesses or depths of these precipitates. These results indicate that the observation of precipitates in crystalline membranes requires some care.     

Comparative study of lithium fluoride and graphite by atomic force microscopy (AFM)

The atomic force microscope (AFM) offers the possibility to image the topography of insulating as well as conductive surfaces. Highly oriented pyrolytic graphite (HOPG) was chosen as an example for a layered material and compared to single crystalline lithium fluoride (LiF). Both materials are easily prepared and inert at ambient pressure. Furthermore they are well characterized by Helium atom scattering experiments and other techniques. On HOPG atomic resolution has been achieved. Distortions can be observed which we interpret as a frictional effect. In addition we performed large area scans where we seldomly observed dislocations. For the first time we present measurements on LiF, showing steps of one unit cell height. On larger areas the surface of LiF showed terraces, separated by steps of variable heights, ranging from a few ångströms to 100 Å. We used a static method to get information about the distance dependence of the force between lever and sample. By slowly expanding and retracting the sample piezo and simultaneous measurement of the lever deflection, plots were recorded, showing the force as a function of sample position. The results were compared with theoretical calculations. We could determine the tip radius and found differences between LiF and HOPG being characteristic for the samples.     

双振镜点扫描三维形貌测量系统

针对远距离大尺寸物体三维形貌测量的要求,设计并实现了基于激光三角法原理的双振镜点扫描三维形貌测量系统。介绍了系统的结构和测量原理,并对系统进行建模,推导了被测物体表面测量点的三维坐标计算公式。构建了原型系统,利用专用的二维平面靶标在测量空间内自由摆放数次,根据靶标上特征点及其像点间的位置关系,以坐标系转换为基础实现了测量系统结构参数的现场标定。利用李萨如图扫描模式分别对靶标平面、已知直径的球面及石膏像自由曲面进行了测量实验。结果表明,测量距离1m处,测量点到拟合平面的距离误差均值为0.70mm,球面直径测量误差均值为0.51mm;测量距离10m处,测量点到拟合平面的距离误差均值为17.85mm。该系统可用于远距离大尺寸物体表面的三维形貌测量。     

Development of high-resolution field emission scanning electron microscopy with multifunctions for chargeless observation of nonconducting surface

We have developed a fully digital field emission scanning electron microscope (FE-SEM) with multifunctions to compensate the charging up of nonconducting surfaces. High-voltage observation, minimum electron dose, variable scanning speed, averaging, integration, tuning of surface potential, and cyclotron movements of secondary electrons have been achieved. This FE-SEM was successfully applied to observe resist, diatomaceous earth, aluminum oxide, and zeolite surfaces. The accelerating voltage is changeable in a range from 0.5 to 30 kV, and the probe current on the sample can be varied from 2×10^-9 to l×10^-13A to supply optimum electron dose. By using a snorkel- type, strongly excited objective lens (OL) immersing the samples in the magnetic field, the secondary electrons are extracted from the sample. For guiding electrons into the built-in lens-type secondary electron detector (SED), newly developed accelerating and retarding electrodes are installed in the OL to tune the surface potential. Furthermore, this FE-SEM can select 10 scan speeds, and the averaging and integration of secondary electron image signals are possible under every selected scan speed.     

Simple procedures for the transfer of grid images onto glass coverslips for the rapid relocation of cultured cells

Procedures are described whereby electron microscope-grid images may be transferred onto the surface of glass coverslips. By using coordinate grids, patterns are obtained that are ideal for the rapid relocation of cultured cells. Two basic procedures are presented. In the first, metal is evaporated, in a vacuum unit, onto glass coverslips carrying electron microscope grids to produce an exposed, glass replica of the grid pattern. The grid pattern is then etched in the glass surface with hydrofluoric acid and the metal subsequently dissolved away. In the second method silicone monoxide is evaporated onto coverslips carrying a ‘negative grid’ or a negative grid image, to produce a pattern directly visible in the phase contrast or interference microscope. Modifications for scanning electron microscopy are also described.