Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Analysis of FIB‐induced damage by electron channelling contrast imaging in the SEM

We have investigated the Ga⁺ ion‐damage effect induced by focused ion beam (FIB) milling in a [001] single crystal of a 316 L stainless steel by the electron channelling contrast imaging (ECCI) technique. The influence of FIB milling on the characteristic electron channelling contrast of surface dislocations was analysed. The ECCI approach provides sound estimation of the damage depth produced by FIB milling. For comparison purposes, we have also studied the same milled surface by a conventional electron backscatter diffraction (EBSD) approach. We observe that the ECCI approach provides further insight into the Ga⁺ ion‐damage phenomenon than the EBSD technique by direct imaging of FIB artefacts in the scanning electron microscope. We envisage that the ECCI technique may be a convenient tool to optimize the FIB milling settings in applications where the surface crystal defect content is relevant.     

Rising the signal-to-noise ratio in X-ray spectra of femtosecond laser-produced plasmas using the “mean-median” algorithm

As femtosecond laser pulses increase in intensity and decrease in duration, interaction between the focused laser radiation and a substance is followed by a sharp increase in the intensity of fast-electron, ion, and electromagnetic noise generation. In turn, the noise signals of X-ray detectors grow in amplitude and the signal-to-noise ratio in recording X-ray spectra of multiply charged ions approaches unity. A significant excess of the noise level over the useful signal is observed in plasmas generated by laser pulses with a power density of ≥10¹⁷ W/cm². The most powerful effect of the above factors is exerted on X-ray spectra recorded by such electromagnetic equipment as CCD-based detectors, photoelectron amplifiers, etc. A new “mean-median” algorithm is described, with which it is possible to considerably increase the signal-to-noise ratio of CCD detectors used to measure X-ray spectra of femtosecond laser-produced plasma.     

Enhancing image contrast of carbon nanotubes on cellular background using helium ion microscope by varying helium ion fluence

Carbon nanotubes (CNTs) have become an important nano entity for biomedical applications. Conventional methods of their imaging, often cannot be applied in biological samples due to an inadequate spatial resolution or poor contrast between the CNTs and the biological sample. Here we report a unique and effective detection method, which uses differences in conductivities of carbon nanotubes and HeLa cells. The technique involves the use of a helium ion microscope to image the sample with the surface charging artefacts created by the He⁺ and neutralised by electron flood gun. This enables us to obtain a few nanometre resolution images of CNTs in HeLa Cells with high contrast, which was achieved by tailoring the He⁺ fluence. Charging artefacts can be efficiently removed for conductive CNTs by a low amount of electrons, the fluence of which is not adequate to discharge the cell surface, resulting in high image contrast. Thus, this technique enables rapid detection of any conducting nano structures on insulating cellular background even in large fields of view and fine spatial resolution. The technique demonstrated has wider applications for researchers seeking enhanced contrast and high‐resolution imaging of any conducting entity in a biological matrix – a commonly encountered issue of importance in drug delivery, tissue engineering and toxicological studies.     

Quasicrystalline Materials

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze-fractured, freeze-dried cultured cells at the ～0.5 μm spatial resolution of the Cameca IMS-3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H^—, C^—, O^—, Na⁺, Mg⁺, CN^—, P^—, S^—, Cl^—, K⁺ and Ca⁺ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.     

A source of gas, vapor, metal, and carbon ions based on a low-pressure hollow-cathode discharge

A compact source of gas, vapor, metal, and carbon ions based on a cold-hollow-cathode reflective discharge has been developed, in which a 6-mm-diameter flat target (Cu, Mo, W, C) is installed on the bottom of the cold cathode insulated from it. The density of the ion flow from cathode plasma reaches 100 mA/cm² at an accelerating voltage of up to 10 kV and a discharge current of 0.2-0.5 A. Vapors produced during ion sputtering of the target are ionized in the cathode and anode cavities. A beam containing ions of the plasma-producing gas and vapor is extracted throug h the channel in the reflector cathode. A fraction of the vapor of the sputtered target, the flow of which is sufficient for growing layers at a rate of ∼0.03 nm/s at a distance of 10 cm from the emission channel under the action of an ion beam, is extracted together with ions. The fraction of metal ions in the extracted beam is 0.05-0.10. The total current of the ion beam is 20-30 mA.     

Tribological properties of nickel-base nanocrystalline coatings sputter-deposited on austenitic stainless steel

500 nm-thick films are deposited on austenitic stainless steel by neutral (Ar⁺) or reactive (N⁺) ion beam sputtering of Ni or NiTi targets, with (or without) high energy 160 keV-Ar⁺ ion beam assistance. Most of the time the coatings are nanocrystalline and induce a large (excellent in some conditions) increase of the wear resistance. Only Ar⁺ ion beam sputtering of a NiTi target gives an amorphous deposit which does not improve the substrate tribological properties. The hardness and wear resistance of ion beam assisted films are larger than those obtained with non-ion beam assisted coatings. The presence of a hard TiN phase inside a ductile Ni phase, of grains with preferential orientation beneficial to slip, as well as film densification are the main factors which increase the wear resistance. The best results are obtained when the structure is composed of two phases, Ni and TiN. The TiN phase strengthens the already good tribological Ni properties and the Ni ductility induces mechanical accommodation during the friction process.     

Edge detection,three-dimensional cell boundary reconstruction and volume and surface area estimation from differential interference contrast images

Three-dimensional (3-D) cell morphology is important for the understanding of cell function and can by quantified in terms of volume and surface area. Differential interference contrast (DIC, or Nomarski) imaging can enable cell edges to be clearly visualized in unstained tissue due to the slight difference in refractive index between aqueous media and cytoplasm. DIC is affected in only one direction - the direction of the optical shear. A 1-D edge detector was used in that direction with a scale length equal to that of an in-focus edge to highlight cell boundaries. By comparison with the signal from the edge detector on an out-of-focus slice, the in-focus slices could be segmented and, after noise suppression, cell outlines obtained. A voxel paradigm was used to calculate cell volume and differential geometry was used for surface area estimation. We applied this approach to obtain 3-D dimensional information by optical sectioning of motile Amoeba proteus.     

Tribological properties of nitrogen ion implanted steel

Nitrogen ions (N⁺) with three different doses were implanted on the AISI 304 LN steel samples under high vacuum at room temperature. Dose dependent morphological and structural changes were observed in the specimen. Structural changes were triggered by formation of nitrides; irradiation induced surface segregation of carbon and deposition of amorphous carbon (a-C) by the cracking of hydrocarbons during implantation from oil diffusion pump. Morphological and structural changes were found to influence nano-mechanical and tribological properties of ion implanted surfaces. The nano-indentation hardness was found to increase to 10.26 GPa with highest N⁺ ion dose due to formation of surface nitrides and amorphous carbon. Frictional force was found to decrease with increase in N⁺ ion dose and a minimum value of 0.078 N was obtained at higher dose presumably due to the formation of amorphous graphite like phase. In addition, amorphous diamond like carbon on the implanted surface can be contributing facts for high hardness. At higher dose, both deformation induced damage and wear rate (2.4 × 10^?7 mm³/Nm) were found to be minimum.     

The evolution of indium precipitation in gallium focused ion beam prepared samples of InGaAs/InAlAs quantum wells under electron beam irradiation

Gallium ion (Ga⁺) beam damage induced indium (In) precipitation in indium gallium arsenide (InGaAs)/indium aluminium arsenide (InAlAs) multiple quantum wells and its corresponding evolution under electron beam irradiation was investigated by valence electron energy loss spectroscopy (VEELS) and high-angle annular dark-field imaging (HAADF) in scanning transmission electron microscopy (STEM). Compared with argon ion milling for sample preparation, the heavier projectiles of Ga⁺ ions pose a risk to trigger In formation in the form of tiny metallic In clusters. These are shown to be sensitive to electron irradiation and can increase in number and size under the electron beam, deteriorating the structure. Our finding reveals the potential risk of formation of In clusters during focused ion beam (FIB) preparation of InGaAs/InAlAs quantum well samples and their subsequent growth under STEM-HAADF imaging, where initially invisible In clusters of a few atoms can move and swell during electron beam exposure.     

Scanning-DLTS,EBIC, and CL Investigations of Diamond Indentations in GaAs p+n Junctions

Diamond indentations have been carried out on Zn-diffused GaAs p⁺n junctions on (100) oriented material. Electron-beam induced current (EBIC) investigations revealed the well-known dislocation slip bands in 〈110〉 directions. Scanning deep level transient spectroscopy (SDLTS) imaging proved a deformation-induced point-defect level at E_y + 0.5 eV, which is preferentially concentrated in the dislocation-free regions between the slip bands rather than within the slip bands. Monochromatic cathodolumines-cence (CL) imaging at 10K using different wavelengths revealed only the dislocation-induced recombination activity but not any point-defect luminescence corresponding to the 0.5 eV level found by SDLTS.     

Freeze-substitution and the preservation of diffusible ions

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (Tl⁺) of Tl⁺-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray micro-analytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K⁺ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used; so far, in the best case, about 67% of the cellular K⁺ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

A microcomputer based digital imaging system for ion microanalysis

A microcomputer based digital imaging system was developed for a Cameca IMS-3f ion microscope permitting real-time digital acquisition of secondary ion images. Image signal-to-noise enhancement results from random noise reduction by real-time ensemble averaging and from a reduction of pattern noise in the charge injection device (CID) array by subtraction of blank frames. Acquired images comprise 244times248 pixel arrays with 8-bit intensity resolution. Images are displayed on a colour monitor in a grey scale or pseudo-colour using one of four programmable lookup tables. Image processing software permits off-line ion image enhancement and manipulation as well as multitechnique digital image correlations. System capability is illustrated by a biological example involving digital imaging studies of Al distribution in osteomalacic bone tissue, including correlative light microscopy and ion microanalysis.     

模糊图像边缘精确定位的滤波算法

图像的边缘是图像最基本也是最重要的特征之一。实际应用中,光学系统离焦和图像传感器噪声源引起图像模糊,导致边缘自动定位算法无法精确确定边缘点。从边缘检测理论出发,根据噪声及图像模糊模型,提出了基于空间域和频域的滤波方法对模糊边缘图像去噪,从而消除模糊图像对于边缘定位的影响。实验表明,各种滤波方法对于边缘点的自动提取都具有一定的效果,离焦模糊和噪声可以得到有效抑制,尤其是频域低通滤波器能够精确地提取出模糊边缘位置。     

Characterization of Si nanocrystals by different TEM-based techniques

Silicon nanocrystals (Si-nc) embedded in SiO₂ matrix and obtained by ion implantation (50 keV, 1.0×10¹⁷ Si/cm²) were characterized by means of three different transmission electron microscopy (TEM) techniques: Dark Field (DF), Scanning TEM Annular Dark Field (STEM-ADF) and Z contrast. The strengths and weaknesses of each technique for the characterization of the Si-nc were evaluated and discussed. DF imaging, which has the best contrast, was chosen to give the average Si-nc size evaluated to 5.6 nm. On the other hand, STEM-ADF, which is only sensitive to the crystalline phase, provided an evaluation of the Si-nc density of 3.27×10¹⁷ nc/cm³. Finally, comparison between the STEM-ADF and Z contrast imaging permitted to evaluate the amorphous phase remaining after the annealing to around 12%.     

基于CNN的十字像中心检测

十字线中心检测是反射法测量透镜中心偏的重要组成部分,十字中心的检测精度决定了透镜中心偏的测量精度。针对边缘不规则、对比度差、信噪比低的图像,提出了基于深度卷积神经网络的十字线中心检测算法。算法的思想是,卷积神经网络可以在一定程度上解决传统算法局限于提取十字像边缘直线和角点特征的问题,实现对十字像整体特征的识别与定位,这可以相对减小图像噪声对十字像中心定位的影响,从而实现在图像质量比较差的情况下对十字像中心准确定位。实验结果表明,提出的算法能够在图像边缘不规则、对比度差、信噪比低等的条件下比较精确得到十字线中心点。     

Ion implantation and surface modification in tribology

Ion implantation is a vacuum process by which virtually any element can be injected into the surface regions of a solid target. Current interest in materials technology and recent developments in ion implantation machines have resulted in ion-induced surface property changes in a variety of different fields including tribology. Large changes in friction coefficient (up to ± 60%) have been recorded on steel surfaces implanted with such ions as Pb⁺ and Sn⁺. Implantation of boron, nitrogen and molybdenum reduces wear by more than a factor of 10, from measurements with a pin-and-disc machine. An outline is given of the scope and application of ion implantation and the results evaluated in the context of the testing methods used. Examples are given of some present and future applications of ion implantation to tribology.     

太赫兹功率探测器高灵敏度调制解调系统设计

针对太赫兹被动成像系统前端功率探测器内部引入的低频噪声（1/f噪声）对成像精度的影响较为明显的问题,设计了一种高灵敏度调制解调系统来降低低频噪声对成像的干扰。通过控制外部信号源使功率探测器在正常工作与饱和状态之间快速切换,实现对目标信号的调制,从而将目标信号的频率调制到高于功率探测器引入的低频噪声频段以便将低频噪声滤除,进而降低该低频噪声对后续成像质量的影响。通过MATLAB软件仿真,可模拟出信噪比(SNR)为31.17 dB的功率探测器输出信号,同时通过对目标信号调制解调,目标信号的信噪比被提高到了268.13 dB。通过对所设计的高灵敏度调制解调系统实际测试,可得目标信号的信噪比由4.22 dB提高到了301.88 dB。研究结果表明,该高灵敏度调制解调系统能有效降低太赫兹探测器内引入的低频噪声对后续成像质量的影响。