Deconvolution of scanning electron microscopy images

This paper describes a method of removing blurs in scanning electron microscopy (SEM) images caused by the existence of a finite beam size. Although the resolution of electron microscopy images has been dramatically improved by the use of high-brightness electron guns and low-aberration electron lenses, it is still limited by lens aberration and electron diffraction. Both are inevitable in practical electron optics. Therefore, a further reduction in resolution by improving SEM hardware seems difficult. In order to overcome this difficulty, computer deconvolution has been proposed for SEM images. In the present work, the SEM image is deconvoluted using the electron beam profile estimated from beam optics calculation. The results show that the resolution of the deconvoluted image is improved to one half of the resolution of the original SEM image.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy,scanning electron microscopy,and transmission electron microscopy

The polyethylene glycol (PEG) embedding and subsequent deembedding method was applied to the observation of general tissues in scanning electron microscopy (SEM). Resulting SEM images were of high quality. It was demonstrated that intermicroscopic correlation of images between light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) is easily and reliably done by means of the PEG method. In particular, the exact correlation of immuno-LM with SEM is shown to be of potential value.     

Electron beam blanking systems

Electron beam blanking in the scanning electron microscope (SEM) by deflection over a chopping aperture is reviewed. The first part is concerned with electron beam deflection structures and driving methods, the second part with electron optics of deflection blanking systems in the SEM.     

扫描电子显微镜在粒度分析中的应用

扫描电子显微镜(SEM)是利用聚焦极细的电子束作为照明源,以光栅状扫描方式照射到试样表面,并以入射电子与试样相互作用所产生的信息来进行成像的。采用扫描电子显微镜对收集在微孔滤膜上的颗粒进行分析,不仅可以观察微小颗粒的表面形貌,还可以与能谱仪配合进行颗粒粒径及数量的测量与统计,测试准确度高,因而在粒度分析领域具有不可替代的作用。主要介绍扫描电子显微镜在粒度分析中的应用。     

Nanotip electron gun for the scanning electron microscope

Experimental nanotips have shown significant improvement in the resolution performance of a cold field emission scanning electron microscope (SEM). Nanotip electron sources are very sharp electron emitter tips used as a replacement for the conventional tungsten field emission (FE) electron sources. Nanotips offer higher brightness and smaller electron source size. An electron microscope equipped with a nanotip electron gun can provide images with higher spatial resolution and with better signal-to-noise ratio. This could present a considerable advantage over the current SEM electron gun technology if the tips are sufficiently long-lasting and stable for practical use. In this study, an older field-emission critical dimension (CD) SEM was used as an experimental test platform. Substitution of tungsten nanotips for the regular cathodes required modification of the electron gun circuitry and preparation of nanotips that properly fit the electron gun assembly. In addition, this work contains the results of the modeling and theoretical calculation of the electron gun performance for regular and nanotips, the preparation of the SEM including the design and assembly of a measuring system for essential instrument parameters, design and modification of the electron gun control electronics, development of a procedure for tip exchange, and tests of regular emitter, sharp emitter and nanotips. Nanotip fabrication and characterization procedures were also developed. Using a "sharp" tip as an intermediate to the nanotip clearly demonstrated an improvement in the performance of the test SEM. This and the results of the theoretical assessment gave support for the installation of the nanotips as the next step and pointed to potentially even better performance. Images taken with experimental nanotips showed a minimum two-fold improvement in resolution performance than the specification of the test SEM. The stability of the nanotip electron gun was excellent; the tip stayed useful for high-resolution imaging for several hours during many days of tests. The tip lifetime was found to be several months in light use. This paper summarizes the current state of the work and points to future possibilities that will open when electron guns can be designed to take full advantage of the nanotip electron emitters.     

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.     

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.     

Atmospheric scanning electron microscopy and its applications for biological specimens

Scanning electron microscopy in ambient conditions (Air‐SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air‐SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)‐SEM. Sharpness was lower than that of FE‐SEM, but foot process of podocytes was observed at high magnification. Air‐SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air‐SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air‐SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.     

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.     

Transient of scanning electron microscopic images for a buried microstructure in insulators

We clarify the transient process and its mechanism of scanning electron microscope (SEM) images of a trench microstructure buried in insulators. First, interface charges of primary electrons trapped on the trench are derived from the charging model of a capacitor considering the electron beam induced current, and the surface potential is therefore assumed. The SEM signal current is then determined from its simplified relation with the surface potential. Calculated profiles of the secondary electron (SE) signal current and their time-evolution behaviors can well fit the transient of the experimental SEM images. Results show that the variation of the surface potential due to the transient interface charges and the effect of SE redistribution result in transients of the SEM imaging signal and the image width of the buried trench.     

Cathodoluminescence attachment to an electron probe instrument

A simple cathodoluminescence (CL) attachment to a JEOL JSM-840 scanning electron microscope (SEM) is described. It is based on a Si photodiode mounted inside the SEM chamber. The current amplifier of a backscattered electron detector unit, which is a standard feature of this SEM, is used to amplify and display the CL images. Examples of CL panchromatic (integral) images of GaAs, InP, and ZnS crystals are presented.     

An approach to the reduction of hydrocarbon contamination in the scanning electron microscope

The deposition of electron beam-induced specimen contamination in both the transmission (TEM) and scanning electron microscopes (SEM) has remained a problem since the beginning of these forms of microscopy. Generally, sources of SEM contamination can be attributed to one or a combination of three major contributors: (1) the pumping system; (2) outgassing of other internal SEM component parts (i.e., specimen stage, stage lubricants, O-rings, etc.), or (3) the sample (including its preparation and handling). Generally, because of the nature of SEM, specimen contamination can be minimized but is difficult to avoid fully. This work outlines three approaches taken with instruments at NIST to reduce the deposition of contamination in high-resolution cold-field emission SEMs. With some modification these techniques could be applied to any SEM. These approaches have been in successful operation for several years, resulting in a reduction in electron beam-induced hydrocarbon contamination.     

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.     

Image sharpness measurement in scanning electron microscopy—part II

A method for qualitative and quantitative analysis of scanning electron microscope (SEM) images forthe determination of sharpness is presented in this paper. Described is a procedure for qualitative analysis based on a software program called SEM Monitor that can be applied to research or industrial SEMs for day-to-day performance monitoring. The idea is based on the fact that, as the electron beam scans the sample, the low-frequency changes in the video signal show information about the larger features and the high-frequency changes give data on finer details. The image contains information about the primary electron beam and about all the parts contributing to the signal formation in the SEM. If everything else is kept unchanged, with a suitable sample, the geometric parameters of the primary electron beam can be mathematically determined. An image of a sample, which has fine details at a given magnification, is sharper if there are more high frequency changes in it. In the SEM, a better focused electron beam yields a sharper image, and this sharpness can be measured. The method described is based on calculations in the frequency domain and can also be used to check and optimize two basic parameters of the primary electron beam, the focus, and the astigmatism.     

Real image resolution of SEM and low-energy SEM and its optimization: distribution width of the total surface emission

The real point resolution of an SEM image is treated in a two-dimensional model where the decisive quantity is the root-mean-square distance of the emitted electron from the pixel centre. This quantity is computed taking into account the direct illumination of the specimen surface by the primary spot the dimensions of which are given by the electron optical column and the indirect illumination from a virtual source of the backscattered electrons in the specimen depth the properties of which depend on the specimen. Because the second order movement of the full distribution is considered here instead of some measure of the central narrow peak of secondaries, the backscattered electron influence significantly deteriorates the resulting resolution. Reasonable approximations regarding both contributions, particularly the Monte-Carlo modelling of the backscattering process by algorithms providing acceptable results down to 1 keV and the approximate relations describing the secondary and backscattered electron emission again down to 1 keV, enabled us to bring the considerations up to numerical results for some typical instruments, namely a "cheap" SEM, a "top" SEM and a low-energy SEM (LESEM) adapted from the cheap SEM by using the cathode lens. The optimum landing energy providing the best point resolution, computed for the individual chemical elements, falls into the range 1 to 5 keV for the cheap SEM and it remains around 1 keV (with some uncertainty caused by the approximations mentioned) for both the top SEM and LESEM. Similarly, the real resolution for the elements ranges between 16 and 45 nm when the cheap SEM with a 3.4 nm nominal spotsize at 30 keV is used, between 3.5 and 9 nm for the 0.7 nm top SEM and between 5.5 and 7 nm for the cheap SEM adapted to LESEM.     

Measurement technique for the incident electron current in secondary electron detectors and its application in scanning electron microscopes.

A measurement technique for incident electron current in secondary electron (SE) detectors, especially the Everhart-Thornley (ET) detector, based on signal-to-noise ratio (SNR), which uses the histogram of a digital scanning electron microscope (SEM) image, is described. In this technique, primary electrons are directly incident on the ET detector. This technique for measuring the correlation between incident electron current and SNR is applicable to the other SE detectors. This correlation was applied to estimate the efficiency of the ET detector itself, to evaluate SEM image quality, and to measure the geometric SE collection efficiency and the SE yield. It was found that the geometric SE collection efficiency at each of the upper and lower detectors of a Hitachi S-4500 SEM was greater than 0.78 at all working distances.     

Observation of atomic steps on single crystal surfaces by a commercial scanning electron microscope.

Atomic steps on (111) and (100) crystal surfaces of Pt were observed using a commercial scanning electron microscope (SEM) in secondary electron mode. By comparing the SEM images and those by reflection electron microscopy (REM), the observed contrast was confirmed to be that from atomic steps on crystal surfaces. The contrast mechanism is briefly discussed. One application of this imaging technique is also shown.     

Practical method for high-resolution imaging of polymers by low-voltage scanning electron microscopy

Morphologic characterization of polymers by scanning electron microscopy (SEM) is often made difficult by their sensitivity to electron beam damage. We describe here a specimen preparation method for the imaging of polymer blends by low-voltage SEM (LV-SEM) that improves their stability in the electron beam and hence facilitates focusing and recording of high magnification images. Its application to nanosized core-shell latexes embedded in a polymethylmethacrylate matrix and semi-crystalline polypropylene/ethylene-propylene rubber blends is discussed.     

Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

Scanning electron microscopy of rust haustoria

The three-dimensional structure of rust haustoria has been studied by scanning electron microscopy (SEM). Rust infected leaves were fixed and embedded in Spurr's resin as for transmission electron microscopy (TEM). The cut surface of blocks containing embedded material was etched with sodium ethoxide and examined in the SEM.