Automatic measurement of scanning beam diameter using an on-line digital computer

A method for the measurement of electron probe beam diameter by digital image processing has recently been published. The purpose of the present report is to describe the development of an automatic system for beam diameter measurement. To complete this system, a method based on a theory which combines automation and high-resolution conditions is proposed. In practice, the beam diameter is measured from the STEM image of a crystalline hole in a gold thin film, utilizing an on-line computer system equipped with newly developed digital processing programs linked to a SEM in the transmission mode. The functions of the programs include statistical processing, matching, noise removal, interpolation, selection, and rotation. By combining these functions, the scanning beam diameter is accurately measured, in spite of difficulties, under most electron microscope operating conditions. The user simply appoints the edge included in the STEM image.     

Observation of dislocations and microplasma sites in semiconductors by direct correlations of STEBIC,STEM and ELS

A high voltage electron microscope, equipped with scanning transmission (STEM) attachment, electron beam induced conductivity (EBIC) facilities, and electron energy loss spectrometer (ELS), has been used to investigate semiconductor devices. The capability of STEM to produce, simultaneously or sequentially, conductive and transmission images of the same specimen region, which can also be ELS analysed, is exploited in order to establish direct and unambiguous correlations between EBIC and STEM images of defective regions (dislocations and microplasma sites) in silicon devices. The results obtained are discussed in terms of correlations, resolution, contrast, and radiation damage; in addition, a comparison is made between this method and the other correlation methods based on EBIC/SEM (scanning electron microscope) and TEM (transmission electron microscope).     

Characterization of precipitates size distribution: validation of low‐voltage STEM

The size distribution of second phase precipitates is frequently determined using conventional transmission electron microscopy (CTEM). However, other techniques, which present different advantages, can also be used for this purpose. In this paper, we focus on high angle annular dark field (HAADF) in TEM and scanning TEM (STEM) in scanning electron microscopy (SEM) imaging modes. The mentioned techniques will be first described, then compared to more conventional ones for the measurement of carbides size distribution in two FeCV and FeCVNb model alloys. This comparative study shows that STEM in SEM, a technique much easier to undertake compared to TEM, is perfectly adapted for size distribution measurements of second phase particles, with sizes ranging between 5 and 200 nm in these systems.     

The conversion of a field-emission scanning electron microscope to a high-resolution,high-performance scanning transmission electron microscope,while maintaining original functions

Recently, the reliability of field-emission electron guns has increased. In addition, the cost of computer systems for on-line processing has dropped. Hence, we should now consider the use of scanning transmission electron microscopy (STEM) for routine work, especially, in the field of biology where one may expect to utilize digital image processing techniques. An STEM has been constructed, without disturbing the original functions, by converting a commercial scanning electron microscope equipped with a fieldemission gun. The STEM is generally operated at accelerating voltage 30 kV, focal length 7.5 mm, and beam current 1?2 × 10^?10 A. Several improvements have been incorporated for removing the effects of vibration, contamination, and stray magnetic fields. Also, an adjustable detector aperture was utilized. The modified instrument was connected to an on-line digital image processing system for utilizing the information obtained from STEM images. The advantages of the modified system were studied from various viewpoints.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

First experimental test of a new monochromated and aberration-corrected 200 kV field-emission scanning transmission electron microscope

The first 200 kV scanning transmission electron microscope (STEM) with an imaging energy filter, a monochromator and a corrector for the spherical aberration (Cs-corrector) of the illumination system has been built and tested. The STEM/TEM concept with Koehler illumination allows to switch easily between STEM mode for analytical and TEM mode for high-resolution or in situ studies. The Cs-corrector allows the use of large illumination angles for retaining a sufficiently high beam current despite the intensity loss in the monochromator. With the monochromator on and a 3 microm slit in the dispersion plane that gives 0.26 eV full-width at half-maximum (FWHM) energy resolution we have obtained so far an electron beam smaller than 0.20 nm in diameter (FWHM as measured by scanning the spot quickly over the CCD) which contains 7 pA current and, according to simulations, should be around 0.12 nm in true size. A high-angle annular dark field (ADF) image with isotropic resolution better than 0.28 nm has been recorded with the monochromator in the above configuration and the Cs-corrector on. The beam current is still somewhat low for electron energy-loss spectroscopy (EELS) but is expected to increase substantially by optimising the condenser set-up and using a somewhat larger condenser aperture.     

Secondary electron imaging as an aid to STEM microanalysis

Secondary electron (SEM mode) imaging in a scanning transmission electron microscope (STEM) has been utilized as an aid to the STEM microanalysis of second phase particles in an Alloy 800 foil by identifying particles at the foil surface facing the incident beam and energy dispersive X-ray detector. Such particles were optimally situated for microanalysis because possible deleterious effects from beam spreading and matrix absorption were eliminated. This was demonstrated by orienting the sample to analyze the particles on the surface of the foil facing away from the beam and X-ray detector, and subsequently, for comparison, reorienting the sample to reanalyze the particles with the foil surface facing beam and detector. Theoretical calculations of the magnitude of the relative change in particle X-ray signal between the two orientations were in good agreement with the experimental observations. SEM imaging also provided surface reference points for parallax-effect measurements of local foil thickness and particle depth within the foil.     

An electron beam lithography and digital image acquisition system for scanning electron microscopes

A low‐cost microcontroller based control and data acquisition unit for digital image recording of scanning electron microscope (SEM) images and scanning electron microscope based electron beam lithography (EBL) is described. The developed microcontroller low‐level embedded software incorporates major time critical functions for image acquisition and electron beam lithography and makes the unit an intelligent module which communicates via USB with the main computer. The system allows recording of images with up to 4096 × 4096 pixel size, different scan modes, controllable dwell time, synchronization with main power frequency, and other user controllable functions. The electron beam can be arbitrary positioned with 12‐bit precision in both dimensions and this is used to extend the scanning electron microscope capabilities for electron beam lithography. Hardware and software details of the system are given to allow its easy duplication. Performance of the system is discussed and exemplary results are presented.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

检测纳米微粒粒径方法的研究

用光子相关法、原子力显微镜和扫描电镜三种测试方法测定了同一标准样品的粒径，比较了三种测试方法在纳米粒径检测方面的特点。光子相关法给出纳米微粒的平均粒径和多分散系数，而原子力显微镜和扫描电镜在测定粒径的同时直接观察到微粒的外貌。     

微纳尺度操作的系统设计

微纳尺度操作是当前先进制造技术领域的一个重要发展方向。为了实现在亚微米及纳米尺度下进行实时观测下的操纵,设计了一种基于SEM的微纳操作系统,本系统由数字式环境扫描电子显微镜系统、精密工件台、微纳操作机构和计算机系统组成,采用数字式环境扫描电子显微镜作为微纳操作的观测器,在SEM的样本室内安装微纳操作臂,计算机系统控制微纳操作臂,使其在SEM的实时观测下对放置在精密工件台上的样本进行操纵。实验结果证明本系统能够适于在各种环境下进行微纳尺度操作,具有很强的实用性。     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.     

Optimization of STEM tomography acquisition — A comparison of convergent beam and parallel beam STEM tomography

In this paper two imaging modes in a state-of-the-art scanning transmission electron microscope (STEM) are compared: conventional STEM with a convergent beam (referred to as nanoprobe) and STEM with a parallel beam (referred to as microprobe). The effect and influence of both modes with respect to their depth of field are investigated. Tomograms of a human white blood cell (hemophagocytes) are acquired, aligned, and evaluated. It is shown that STEM using a parallel beam produces tomograms with fewer distortions and artifacts that allows resolving finer features. Microprobe STEM tomography is advantageous especially in life science, when semi-thin sections (approximately 0.5 μm thick) of biological samples are imaged at relatively low magnification with a large field of view.     

Characterization of the μ and P phase precipitates in the CMSX‐4 single crystal superalloy

A combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning‐transmission electron microscopy (STEM) using high‐angle annular‐dark‐field (HAADF) imaging, focussed ion beam‐ scanning electron microscopy (FIB‐SEM) tomography, selected area electron diffraction with beam precession (PED), as well as spatially resolved energy‐dispersive X‐ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS), was used to investigate topologically close‐packed (TCP) phases, occurring in the CMSX‐4 superalloy subjected to high temperature annealing and creep deformation. Structural and chemical analyses were performed to identify the TCP phases and provide information concerning the compositional partitioning of elements between them. The results of SEM and FIB‐SEM tomography revealed the presence of merged TCP particles, which were identified by TEM and PED analysis as coprecipitates of the μ and P phases. Inside the TCP particles that were several micrometres in size, platelets of alternating μ and P phases of nanometric width were found. The combination of STEM‐HAADF imaging with spatially resolved EDS and EELS microanalysis allowed determination of the significant partitioning of the constituent elements between the μ and P phases.     

Digital acquisition and processing of electron micrographs using a scanning transmission electron microscope

A digital acquisition system that collects multichannel information from a scanning transmission electron microscope (STEM) and its application are described. The hardware comprises (i) single electron counting detectors, (ii) a digital scan generator, (iii) a digital multi-channel on-line processor, (iv) an interface to a minicomputer, and (v) a display system. Experimental results characterizing these components are presented, and their performance is discussed The software includes assembler coded programs for dynamic file maintenance and fast acquisition of image data, a display driver, and FORTRAN coded application programs. The usefulness of digitized STEM is illustrated by a variety of biological applications.     

Thick specimens in the CEM and STEM. Resolution and image formation.

A theory of resolution and image formation is presented for thick amorphous specimens in transmission electron microscopes. Eight modes of operation are considered, four in the scanning transmission electron microscope (STEM) and four in the conventional electron microscope (CEM). A thick specimen is defined here as one in which the resolution of detail is limited by plural scattering of the electron beam. In practice this includes films on the order of a micron in thickness. An analytic theory of plural incoherent scattering is developed which is general with respect to material and beam voltage. The theory gives the distribution of elastically scattered electrons as a function of transverse coordinate and angles, and is directly applicable to optical systems. The theory applies to all thicknesses normally encountered, and includes thin specimens as well as thick specimens. Criteria are proposed for evaluation of the quality of microscope images, and the modulation transfer function is applied to determine some practical estimates of picture quality. The STEM is found to have distinct advantages over the CEM for thick specimens. For a carbon specimen one micron thick a STEM operating in bright field at 90 keV produces an image which is roughly equivalent to that of a CEM operating in bright field at 1 MeV. Improvement can be obtained in the CEM by filtering out eneryg-loss electrons which degrade resolution due to chromatic aberration. This results in a reduction in signal intensity and usable thickness, however.     

Beam spreading and spatial resolution in thick organic specimens

Tomography using a scanning transmission electron microscope (STEM) offers intriguing possibilities for the three-dimensional imaging of micron-thick, biological specimens and assemblies of nanostructures, where the image resolution is potentially limited only by plural elastic scattering in the sample. A good understanding of the relationship between material thickness and spatial resolution is required, with particular emphasis on the competition between beam divergence (a geometrical effect from the converged STEM probe) and beam spreading (an unavoidable broadening due to plural elastic scattering). We show that beam divergence dominates beam spreading for typical embedding polymers beyond the 100-nm thickness range and that minimization of this effect leads to enhanced spatial resolution. The problems are more pronounced in spherical-aberration-corrected instruments where the depth of field is shorter.     

Minority carrier lifetime mapping in the SEM

A method is described for obtaining two-dimensional distributions of minority carrier lifetimes in semiconductor materials for optoelectronic applications. The novel features of the system are: on line analogue calculation of the lifetime from the decay of the cathodoluminescence signal after switching off the SEM electron beam; simultaneous computerized mapping of the signals obtained in this manner in the scanning electron microscope. Typically, it permits establishing a map of 1 mm² with 512 times 512 data points in 10 min for lifetimes down to 3 ns.     

Electron-beam-induced reduction of Fe in iron phosphate dihydrate,ferrihydrite, haemosiderin and ferritin as revealed by electron energy-loss spectroscopy

The effect of high-energy electron irradiation on ferritin/haemosiderin cores (in an iron-overloaded human liver biopsy), its mineral analogue; six-line ferrihydrite (6LFh), and iron phosphate dihydrate (which has similar octahedral ferric iron to oxygen coordination to that in ferrihydrite and ferritin/haemosiderin cores) has been investigated using electron energy-loss spectroscopy (EELS). Fe L_2,3-ionisation edges were recorded on two types of electron microscope: a 200 keV transmission electron microscope (TEM) and a 100 keV scanning transmission electron microscope (STEM), in order to investigate the damage mechanisms in operation and to establish a methodology for minimum specimen alteration during analytical electron microscopic characterisation. A specimen damage mechanism dominated by radiolysis that results in the preferential loss of iron co-ordinating ligands (O, OH and H₂O) is discussed. The net result of irradiation is structural re-organisation and reduction of iron within the iron hydroxides. At sufficiently low electron fluence and particularly in the lower incident energy, finer probe diameter STEM, the alteration is shown to be minimal. All the materials examined exhibit damage which as a function of cumulative fluence is best fitted by an inverse power-law, implying that several chemical and structural changes occur in response to the electron beam and we suggest that these are governed by secondary processes arising from the primary ionisation event. This work affirms that electron fluence and current density should be considered when measuring mixed valence ratios with EELS.     

Scanning reflection image from a solid specimen in the scanning electron microscope with a condenser-objective lens

To achieve the highest resolution in the scanning electron microscope (SEM) or in the scanning transmission electron microscope (STEM), the sample must be mounted in the high-field region of a condenser-objective lens. A secondary electron (SE) image can then be obtained using a collector before the lens. It is also possible to obtain a scanning reflection image by tilting the specimen so that the second half of the condenser-objective lens field deflects the forward-scattered electrons onto the transmission detector beyond the specimen. Experiments were made with an unmodified commercial SEM fitted with a condenser-objective in the upper stage and with a transmission detector, and it was found that the scanning reflection image from a solid sample can provide additional useful information when used in conjunction with the SE image.