Microtomography from limited projections in conventional TEM for 3D reconstruction of an intact cell

It is possible to generate three-dimensional reconstructions of whole, non-sectioned biological cells in conventional TEM using an 80 kV tungsten source. A TEM specimen stage was modified to accommodate a precise single-axis tilting mechanism controlled by a digital stepping motor interfaced to a computer. For image collection, a video camera was optically coupled to the TEM phosphorescent screen, and the video image was digitized by a frame buffer interfaced to a computer. Specimen tilt and projection image collection were fully computer-automated. This microtomography system design could be readily adapted for most TEMs. Image reconstruction was achieved through computation on projection images from limited tilts; typically less than thirty projection images were needed for a coarse 3D reconstruction. The iterative reconstruction algorithm used certain statistical assumptions about the distribution of image gray values. Since microtomography was performed on non-sectioned whole mount cells viewed under an 80 kV electron beam, methods of embedment-free specimen preparation with chemical fixation and extraction were employed. These methods were utilized successfully to permit good image formation of the entire cell mitotic nucleus a few micrometers in thickness. The 3D reconstruction of a single kidney cell mitotic nucleus was carried out and shown to produce a reasonable microtomogram of gross features like the condensed chromosomes.     

Three-dimensional reconstruction of cells from serial sections and whole-cell mounts using multilevel contouring of stereo micrographs

A comprehensive computer-graphics-based system (STERECON) is described for tracing and digitizing contours from individual or stereopair electron micrographs. The contours are drawn in parallel planes within the micrographs. Provision is also made for tracing and digitizing in full three-dimensional (3-D) coordinates in any direction along linear structures such as cytoskeletal elements. The stereopair micrographs are viewed in combination with the contours being traced on a graphics terminal monitor. This is done either by projecting original electron micrograph (EM) negatives onto a screen and optically combining these images with contour lines being drawn on the monitor, or by first digitizing the images and displaying them directly on the monitor along with the contour lines. Prior image digitization allows computer enhancement of the structures to be contoured. Correction and alignment routines are included to deal with variable section thickness, section distortion and mass loss, variations in photography in the electron microscope, and terminal screen curvature when combining projected images with contour lines on the monitor. The STERECON system organizes and displays the digitized data from successive sections as a 3-D reconstruction. Reconstructions can be viewed in any orientation as contour stacks with hidden lines removed; as wire-frame models; or as shaded, solid models with variable lighting, transparency, and reflectivity. Volumes and surface areas of the reconstructed objects can be determined. Particular attention was paid to making the system convenient for the biological user. Users are given a choice of three different stereo-viewing methods.     

Reconstruction from in-line electron holograms by digital processing

The digitized in-line electron holograms recorded in an HB-5 STEM instrument were reconstructed by computer processing in place of optical reconstruction. The important parameters of the holograms, namely the spherical aberration and defocus, can be obtained from the electron Ronchigrams. The reconstructed image shows some improvement in resolution as compared with the STEM bright field image. The main limitations appear to be mechanical vibration, drift of the specimen, the relatively small number of pixels in the images, and the uncertainties in determining the spherical aberration coefficient and defocus value.     

Automatic analysis of G-banded human chromosomes by fast scanning microscope photometry. I. Basic principles and computer control

A high resolution scanning microscope photometry system has been developed for use with standard G-banded chromosome preparations. Various alternative scanning methods and their limitations are reviewed. The computer algorithms which we use are described for the manipulation of the digitized image as produced by the microscope photometer; these include noise reduction and contrast enhancement as well as altering the orientation of the chromosome.     

Two-phase flow in porous media: obtaining sharp digitized images of serial sections for subsequent quantitative analysis

Microscopic studies of the distributions of oil and aqueous phases within the pore space of reservoir rocks, at various relative saturations, are of fundamental importance in understanding migration, distribution, mobilization and recovery of oil in the context of a particular (enhanced) oil recovery method. The most effective approach to use for these studies is three-dimensional computer reconstruction with the data for the three-phase system (rock, oil, aqueous phase) entered by video digitization of a set of serial sections. A novel technique for creating accurate three-phase digitized images from the serial section data is described. By use of appropriate pore cast materials and lighting conditions, three different two-phase video images are created and digitized. In each image, a different one of the three phases in the pore cast is one of the two distinguishable phases. The two best two-phase digitized images are combined to give the three-phase digitized image.     

Scanning image detection (SID) system for conventional transmission electron microscope (CTEM) images

A new image detection system has been developed to display transmission electron microscope (TEM) images on a CRT without a video camera system. Deflection coils placed in both the upper space of an objective lens and in the lower space of the first intermediate lens scan a small electron probe simultaneously. The electrical signal acquired through an improved scintillator and a photomultiplier is synchronized with the scanning signal and displayed in a similar fashion to a conventional scanning TEM (STEM) instrument. A preliminary system using a 100 kV conventional TEM (CTEM) equipped with a hairpin-type electron gun, produced an image with a spatial resolution of 1 nm.     

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.     

A simple computer program to obtain digital pseudo-colored high resolution electron microscopy images

Computer programs which allow one to simulate high resolution electron microscopy images generally deliver the output on a line printer with the overprinting technique. Although this method suffers from poor resolution and limited contrast dynamics, it is still in widespread use. A more effective way is to represent the images in digital form on a color video display, and several solutions have been proposed recently, which often, however, make necessary use of rather expensive pictorial video terminals. In this work we describe a simple program which allows one to represent the output of a multislice program (projected potentials, lattice images, and diffraction patterns) on the display of an inexpensive color video terminal. Useful operations on the image, such as color selection over a wide range, image magnification, and filtering (to enhance specific details of interest) have been implemented. The displayed image can be either photographed from the screen or recorded on a good quality hard copier.     

Similar features in Z bands of both skeletal and cardiac muscle revealed by image enhancement

We have shown previously that the small square (ss) and basket weave (bw) states of the Z band lattice in cardiac and skeletal muscle are related to the contractile state of the muscle. We have used two-dimensional image processing techniques on digitized electron micrographs to enhance the structural features of each projected lattice form in cardiac and skeletal muscle. Four different processing techniques were employed to assess the effect of enhancement artifacts on the resulting Z band images. We observed only slight differences between enhanced images of a particular Z band form produced by the four different techniques. Every enhanced image showed an approximate four-fold symmetry independent of muscle type or Z band lattice form. Each enhanced image showed four cross-connecting Z-filaments which appeared to connect each axial filament to the four nearest axial filaments. In bw images from both cardiac and skeletal muscle, axial filaments had a greater apparent diameter and a greater interaxial filament spacing than in the ss images. In both muscle types, the cross-connecting Z-filaments appeared to overlap half-way between axial filaments in the ss images while the bw images showed no such overlap. These structural features are consistent with a dynamic Z band lattice that participates in muscle contraction.     

Data acquisition system for maximum-likelihood analysis of electron microscopic autoradiographs

EMAMAP is a program for the data acquisition phase of maximum-likelihood analysis of electron microscope autoradiographs. This program is written in C and has been implemented on a Masscomp MC-500 which supports a graphics processor and a digitizing tablet. The image analysis is automated at a low level: the program operator outlines the edges of the structures of interest using the digitizing tablet, while contiguous regions formed by closed contours are automatically filled by the software. The resulting image is compressed for efficient storage by a quadtree encoding technique for which data compression ratios of greater than 25:1 have been achieved. In practical terms this implies that the data from a typical experiment of 50 autoradiographs could be stored on a single floppy disk. The system is currently in use for acquiring actual biological experimental data.     

A simple method for the acquisition of high-quality digital images from analog scanning electron microscopes

A method is described for converting video signals of analog scanning electron microscopes (SEMs) into digital images of high quality. A plug-in card commercially available for personal computers is used for the on-line analog/digital conversion. A Windows application program written by the authors, together with low-level software drivers supplied with the plug-in card, allow digital images to be recorded, to be displayed simultaneously on the computer monitor and to be saved as a file in a standardized format. Compared to conventional photographic images obtained from the SEM camera system, the digital images possess superior sharpness of outline, excellent image definition, diminished noise and well-defined grey-scale tones. This method provides SEM images of high quality for less than $1000 from most older analog SEMs. In addition, the advantages of digital image processing can be applied to analog SEMs, including contrast enhancement, digital filtering and multichannel recording.     

High-resolution imaging of organic pharmaceutical crystals by transmission electron microscopy and scanning moiré fringes

Formulation processing of organic crystalline compounds can have a significant effect on drug properties, such as dissolution rate or tablet strength/hardness. Transmission electron microscopy (TEM) has the potential to resolve the atomic lattice of these crystalline compounds and, for example, identify the defect density on a particular crystal face, provided that the sensitivity of these crystals to irradiation by high-energy electrons can be overcome. Here, we acquire high-resolution (HR) lattice images of the compound furosemide using two different methods: low-dose HRTEM and bright-field (BF) scanning TEM (STEM) scanning moiré fringes (SMFs). Before acquiring HRTEM images of furosemide, a model system of crocidolite (asbestos) was used to determine the electron flux/fluence limits of low-dose HR imaging for our scintillator-based, complementary metal-oxide semiconductor (CMOS) electron camera by testing a variety of electron flux and total electron fluence regimes. An electron flux of 10 e⁻/(Ų s) and total fluence of 10 e⁻/Ų was shown to provide sufficient contrast and signal-to-noise ratio to resolve 0.30 nm lattice spacings in crocidolite at 300 kV. These parameters were then used to image furosemide which has a critical electron fluence for damage of ≥10 e⁻/Ų at 300 kV. The resulting HRTEM image of a furosemide crystal shows only a small portion of the total crystal exhibiting lattice fringes, likely due to irradiation damage during acquisition close to the compound's critical fluence. BF-STEM SMF images of furosemide were acquired at a lower electron fluence (1.8 e⁻/Ų), while still indirectly resolving HR details of the (001) lattice. Several different SMFs were observed with minor variations in the size and angle, suggesting strain due to defects within the crystal. Overall BF-STEM SMFs appear to be more useful than BF-STEM or HRTEM (with a CMOS camera) for imaging the crystal lattice of very beam-sensitive materials since a lower electron fluence is required to reveal the lattice. BF-STEM SMFs may thus prove useful in improving the understanding of crystallization pathways in organic compounds, degradation in pharmaceutical formulations and the effect of defects on the dissolution rate of different crystal faces. Further work is, however, required to quantitatively determine properties such as the defect density or the amount of relative strain from a BF-STEM SMF image.     

TEM纳米尺度石墨标样的制备

透射电镜的长度标尺或公称放大倍数是判断试样中组织细节尺寸的依据,需要应用纳米级长度标样进行校准。本文介绍了一种用石墨制备透射电镜专用纳米尺度标样的方法,对石墨的X射线衍射分析以及对石墨标样的TEM高分辨像和电子衍射分析表明,标样中石墨的高分辨像为（002）晶面的点阵像,其晶面间距为0．342nm,可作为纳米尺度的参照材料。本文对使用纳米尺度标样校正ETEM标尺的基本方法进行了简要讨论。     

Laser scanning phase modulation microscope

We describe the concept and first implementation of an innovative new instrument for quantitative light microscopy. Currently, it provides selective imaging of optical path differences due to birefringence; with further development, it is also possible to selectively image several optical properties, including refractive path differences, optical rotation, and linear and circular dichroism, all with diffraction-limited resolution. An image consists of a 512×512 element array, with each pixel displaying one of 256 grey levels, linearly proportional to the specific optical property being observed. Additionally, conventional brightfield and polarized light microscopy are available, with the accompanying advantages of laser scanning and digital image processing. The microscope consists of three subsystems, representing three distinct technologies. The laser scanning subsystem moves a focused, microspot across the specimen; the output of a photodetector is an electric signal corresponding to a scanned image. The image display subsystem digitizes this signal and displays it as an image on a video monitor. When used in conjunction with a phase modulation feedback loop, the image formed is of the specimen's birefringent retardation or other selected optical property. The digitized images are also available for computer enhancement.     

基于计算机视觉的药片实时检测系统

为了提升药品缺陷检测效率,降低制药企业的生产成本,设计了一款基于计算机视觉的药片实时检测系统。介绍了计算机视觉检测系统的工作原理和硬件结构组成,并在此硬件结构基础上,提出了一种新型计算机图像分割和特征提取方法。实验结果表明,所述算法和系统能够显著提高药片缺陷检测准确度,可以达到99%,计算机视觉检测系统能够完全满足实时性、高识别率等要求。     

Real time computer simulation of electron microscope images with tilted illumination: Grain boundary applications

Computer programs have been developed to simulate electron microscope images from digitized graphically represented model structures. Via a television rate image processing system, these programs allow real time, interactive modification of the microscope objective lens parameters, incident beam inclination, and incident beam energy. In addition to explaining the computational methods, the need for using tilted beam illumination is explored to extend microscope resolution. For this study, the subject of grain boundary imaging is analyzed for a copper σ = 5,36.9°,(310) tilt boundary with a [001] common rotation axis. The Cu {200} lattice spacings of ～1.8Å on both sides of the interface cannot be reliably resolved under axial illumination conditions in a 200 kV microscope. Therefore, either tilted beam modes or higher incident beam energies were explored and the types of image features correlated with atomic position data through the digital frame store system.     

Imaging columns of the light elements carbon, nitrogen and oxygen with sub Angstrom resolution.

It is reported that lattice imaging with a 300 kV field emission microscope in combination with numerical reconstruction procedures can be used to reach an interpretable resolution of about 80 pm for the first time. A retrieval of the electron exit wave from focal series allows for the resolution of single atomic columns of the light elements carbon, nitrogen, and oxygen at a projected nearest neighbor spacing down to 85 pm. Lens aberrations are corrected on-line during the experiment and by hardware such that resulting image distortions are below 80 pm. Consequently, the imaging can be aberration-free to this extent. The resolution enhancement results from increased electrical and mechanical stability of the instrument coupled with a low spherical aberration coefficient of 0.595 + 0.005 mm.     

Digital image acquisition in in vivo confocal microscopy

A flexible system for the real-time acquisition of in vivo images has been developed. Images are generated using a tandem scanning confocal microscope interfaced to a low-light-level camera. The video signal from the camera is digitized and stored using a Gould image processing system with a real-time digital disk (RTDD). The RTDD can store up to 3200 512 times 512 pixel images at video rates (30 images s^?1). Images can be input directly from the camera during the study, or off-line from a Super VHS video recorder. Once a segment of experimental interest is digitized onto the RTDD, the user can interactively step through the images, average stable sequences, and identify candidates for further processing and analysis. Examples of how this system can be used to study the physiology of various organ systems in vivo are presented.     

Digital beam control for fast differential voltage contrast

A new method for voltage contrast enhancement by image subtraction is presented. It is based on a modification of a commercial column automation system that digitizes beam positions and intensity signals in a scanning electron microscope (SEM). The new modulus performs via hardware all the operations of bias control and signal differentiation at each image point, resulting in a minimum acquisition time of 4 μs per point, that is less than 0.3 seconds to obtain a 256 × 256 pixels image with 8 bit gray resolution. The digital handling of intensity data, gives high precision differential images and the control of beam position prevents spatial differentiation effects, enabling noise reduction by time integration of intensity data in each point.     

A method for reconstructing patterns of somatosensory cerebral cortical activity

An interactive graphics package was developed in order to acquire, display, and manipulate images of cerebral cortical autoradiographic data. The primary purpose for development of the system was to reconstruct accurate 2-dimensional maps of the functional activity within the somatosensory cerebral cortex. A Datacube Q-bus graphics module (QVG/QAF-123) was interfaced with the Micro PDP-11/23 to accept a standard RS170 video input signal, and autoradiographs of serial sections (each 20 microns thick) of a cerebral cortex were digitized individually to 768 X 512 X 8 bit resolution. Input look-up tables were used to standardize the autoradiographic data. Boundaries of the somatosensory cortex were entered (with a Summagraphics MM 1201 digitizer), and the image data was stored on disk file (a method of data compression was devised). A method for segmenting the image data for many (sequential) sections was developed that provided arrays from which the maps were generated. Thresholding, histogram equalization, edge detection and edge enhancement, and filters in both the spatial and frequency domains were employed to process the images of the maps. Plots of optical density values along any axis of the maps and gray level histograms of any map region could also be generated. Maps made by the described method are much higher in resolution than those produced by traditional (manual) methods, and permit analysis of the reconstructions in both the frequency and spatial domains.