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.     

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.     

New system for secondary electron detection in variable-pressure scanning electron microscopy

A novel secondary electron detection system combining a two‐stage detector head and a differential pumping system is presented. The detector head consisted of a scintillation Everhart‐Thornley detector and a microsphere plate, separating it from the lower vacuum in the intermediate chamber (below 0.1 mbar). The system was arranged asymmetrically, which should contribute to a lower gas leakage through the plate and a longer life span of the plate. The system offered all the advantages of the scintillator detector in a wide range of gas pressures, from high vacuum to those of the order of 10 mbar, typical of high‐pressure scanning electron microscopy.     

A system for acquiring simultaneous electron energy-loss and X-ray spectrum-images

A compositional imaging system based on simultaneous scanning electron energy‐loss spectroscopy (EELS) and energy‐dispersive X‐ray spectroscopy (EDS) was developed. This system utilizes the combined power of EELS and EDS for quantitative compositional imaging at nanometre resolution. The system is particularly suitable for, but not limited to, biological research, as it simultaneously provides sensitive maps of an element such as Ca or P from EELS and of many other elements from EDS. Degradation of resolution by specimen drift is prevented by correcting for drift during data acquisition, using image cross‐correlation. Several advanced features are implemented for real‐time and/or off‐line quantitative analysis, and the performance of the system is illustrated with practical applications to compositional imaging of cardiac muscle.     

High‐resolution,high‐throughput imaging with a multibeam scanning electron microscope

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single‐beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.     

Scanning electron microscope electron detector with a radial type discrete dynode electron multiplier

A discrete dynode electron multiplier with radial flux of electrons was built and tested in the range of low‐voltage scanning electron microscopy as a backscattered electron detector of topographic contrast. The multiplier collects backscattered electron emitted in a specific range of take‐off angles and over the whole azimuth angular range enabling large solid collection angle. Multipliers with different dynode shapes were studied theoretically with the use of the software for particle optics and three assemblies were built and tested experimentally. The gain estimation, assessment of the type of detected electrons (secondary electron or backscattered electron), imaging the spatial collection efficiency and signal‐to‐noise measurements were performed.     

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.     

ESCA-750型电子能谱仪测控系统改造

依据电子能谱仪的测控原理，设计了ESCA-750型电子能谱仪的计算机测控系统，利用工业级PCI数据采集卡搭建硬件接口平台，采用面向对象的程序设计方法分层分块设计测控软件，实现了仪器的计算机控制与数字化采样，恢复使用长期停用的仪器。该系统采用的软硬件设计方法对旧型电子能谱仪的改造有借鉴作用。     

Electronic image formation in high-voltage TEM by sequential pixel acquisition

We describe a complete hybrid imaging system for the electronic detection and manipulation of high-voltage TEM images. The system includes a recently incorporated two-dimensional beam deflector for dissecting the image into pixels by displacing it in a raster in front of an aperture leading to an electron detector. The characteristics of images thus formed by sequential pixel acquisition and displayed on a CRT are discussed and illustrated. Absorption or thickness profiles are also obtained by displaying the scanned pixel intensity in Y-modulation. Similarly, the displacement of an entire diffraction pattern allows the intensities of individual diffraction spots to be quantitated directly over a wide dynamic range. The direct splitting of the image into electronically controlled pixels can be applied to local mass measurements and to energy-loss analysis by means of a simple magnetic prism that can also provide energy filtering in lieu of a more elaborate electron-optical, image-preserving system.     

Correction of image drift and distortion in a scanning electron microscopy

Continuous research on small‐scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high‐resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift–time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three‐order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high‐resolution electron microscopic system.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

Real time uses of color for electron microscopy via a television-rate digital frame store

An on-line television-rate digital frame store device is utilized to provide color representations of a wide range of electron microscope images and image data. Various types of hardware devices in the frame store coupled with software manipulations via the host computer make rapid image acquisition, modification, measurement, and full-color display possible in real time either from micrographs or directly from an electron microscope. Lookup tables used in conjunction with grey-level image memories can be controlled from a menu display to provide a wide range of color-coding schemes and sequencing. It is also possible to use color graphics overlays and alpha numeric displays along with full-color image displays. This paper will describe many of the recent applications of color developed for electron microscopy studies of materials.     

The effects of space charge on contrast in images obtained using the environmental scanning electron microscope

We present experimental evidence for the existence of a space charge in the environmental scanning electron microscope. Space charge formation is attributed to differences in the mobilities of negative and positive charge carriers in the imaging gas. A model is proposed for the behavior of space charge during image acquisition. The effects of space charge on images acquired using the gaseous secondary electron detector, ion current, and backscattered electron signals are interpreted using the proposed model.     

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.     

Non‐rigid alignment in electron tomography in materials science

Electron tomography is a key technique that enables the visualization of an object in three dimensions with a resolution of about a nanometre. High‐quality 3D reconstruction is possible thanks to the latest compressed sensing algorithms and/or better alignment and preprocessing of the 2D projections. Rigid alignment of 2D projections is routine in electron tomography. However, it cannot correct misalignments induced by (i) deformations of the sample due to radiation damage or (ii) drifting of the sample during the acquisition of an image in scanning transmission electron microscope mode. In both cases, those misalignments can give rise to artefacts in the reconstruction. We propose a simple‐to‐implement non‐rigid alignment technique to correct those artefacts. This technique is particularly suited for needle‐shaped samples in materials science. It is initiated by a rigid alignment of the projections and it is then followed by several rigid alignments of different parts of the projections. Piecewise linear deformations are applied to each projection to force them to simultaneously satisfy the rigid alignments of the different parts. The efficiency of this technique is demonstrated on three samples, an intermetallic sample with deformation misalignments due to a high electron dose typical to spectroscopic electron tomography, a porous silicon sample with an extremely thin end particularly sensitive to electron beam and another porous silicon sample that was drifting during image acquisitions.     

Three‐dimensional optical transfer functions in the aberration‐corrected scanning transmission electron microscope

In the scanning transmission electron microscope, hardware aberration correctors can now correct for the positive spherical aberration of round electron lenses. These correctors make use of nonround optics such as hexapoles or octupoles, leading to the limiting aberrations often being of a nonround type. Here we explore the effect of a number of potential limiting aberrations on the imaging performance of the scanning transmission electron microscope through their resulting optical transfer functions. In particular, the response of the optical transfer function to changes in defocus are examined, given that this is the final aberration to be tuned just before image acquisition. The resulting three‐dimensional optical transfer functions also allow an assessment of the performance of a system for focal‐series experiments or optical sectioning applications.     

电子束焊缝二次电子成像系统设计研究

在研究二次电子特性及成像机理的基础上,设计了二次电子成像系统.其原理是通过同步扫描系统使焊机与显像管中电子束同步光栅扫描,用采集板收集从工件上轰击出来的二次电子,此二次电子信号通过前置放大和视频放大,最后在显像管得到焊缝图像.并且该系统在实验中得到了初步预期的结果.     

Digital image processing: a path to better pictures

Digital image processing can be used to provide enhanced performance in scanning electron column instruments. Improved visualization, reduced specimen damage, and quantization can be achieved. The system described here provides hardware for digitization and storage of multiple images simultaneously, and multimode scan generation. System software provides easy scan control, image digitization, automatic prescale adjust on acquisition, grey scale histogram generation, grey scale compression or expansion, image filtering, smoothing, and random color assignment to grey levels. Image digitization and processing was done using the EG&G ORTEC Image Master Microanalyzer in conjunction with a JEOL 100CX TEMSCAN and a JEOL JSM 35. Examples of image processing of bright and dark field STEM images of biological specimens are shown. An example of X-ray image processing using a two-dimensional filter function to reduce image noise is also shown.     

Local crystal structure analysis with several picometer precision using scanning transmission electron microscopy

We report a local crystal structure analysis with a high precision of several picometers on the basis of scanning transmission electron microscopy (STEM). Advanced annular dark-field (ADF) imaging has been demonstrated using software-based experimental and data-processing techniques, such as the improvement of signal-to-noise ratio, the reduction of image distortion, the quantification of experimental parameters (e.g., thickness and defocus) and the resolution enhancement by maximum-entropy deconvolution. The accuracy in the atom position measurement depends on the validity of the incoherent imaging approximation, in which an ADF image is described as the convolution between the incident probe profile and scattering objects. Although the qualitative interpretation of ADF image contrast is possible for a wide range of specimen thicknesses, the direct observation of a crystal structure with deep-sub-angstrom accuracy requires a thin specimen (e.g., 10 nm), as well as observation of the structure image by conventional high-resolution transmission electron microscopy.     

Automated microscopy system for mosaic acquisition and processing

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide‐field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast‐scanning, multiphoton, confocal microscope fitted with a motorized, high‐precision stage and custom‐developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.