The integration of computers and transmission electron microscopes (TEM) in combination with the availability of computer networks evolves in various fields of computer-controlled electron microscopy. Three layers can be discriminated: control of electron-optical elements in the column, automation of specific microscope operation procedures and display of user interfaces. The first layer of development concerns the computer-control of the optical elements of the transmission electron microscope (TEM). Most of the TEM manufacturers have transformed their optical instruments into computer-controlled image capturing devices. Nowadays, the required controls for the currents through lenses and coils of the optical column can be accessed by computer software. The second layer of development is aimed toward further automation of instrument operation. For specific microscope applications, dedicated automated microscope-control procedures are carried out. in this paper, we will discuss our ongoing efforts on this second level towards fully automated electron tomography. The third layer of development concerns virtual- or telemicroscopy. Most telemicroscopy applications duplicate the computer-screen (with accessory controls) at the microscope-site to a computer-screen at another site. This approach allows sharing of equipment, monitoring of instruments by supervisors, as well as collaboration between experts at remote locations.

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments.

Cryo-electron microscopy is becoming an increasingly powerful tool for solving the structure of protein complexes and has the potential to address problems that cannot be solved using other methods. The field however suffers from several major disadvantages related to the time required to acquire, process and analyze the data and the tedium of using the current prevailing methods. We have for some time been working towards the goal of developing a system that will result in a 3D map of a macromolecular structure automatically and within hours of inserting a specimen into a transmission electron microscope. We propose that these automated methods for data collection and analysis will have a significant impact in transferring the cryo-electron microscopy technology to the general biological community as well as in increasing the volume of data that can be collected during a single session at the microscope.

The Leginon system that we have developed is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low dose conditions and acquiring images at high magnification to either film or a digital camera.

In high-resolution biological electron microscopy, the speed of collection of large numbers of micrographs of the required quality is a rate-limiting step in the overall process of structure determination. Approaches to speed up data collection can be very useful especially in “single molecule” microscopy of large multi-protein and protein-nucleic acid complexes, where many thousands of individual molecular images need to be averaged to determine the threedimensional structure.

Here, we report the development of an automated low-dose image acquisition procedure on a Tecnai 12 electron microscope using the scripting functionality available on the microscope computer. The program that we have developed and implemented, AutoEM, is presently aimed at providing automation capabilities for the user familiar with normal operation of the microscope. However, the user’s participation is relatively minimal, and involves the following steps. After preparing a specimen grid and inserting it into the microscope as is normal for a cryo transfer session, the user simply downloads a Low-Dose alignment file that contains information on the relative beam shifts between the Search, Focus and Exposure modes that would normally be employed in a manual low-dose data collection experiment.

Recently, SEM (Scanning Electron Microscope) and the other observation devices are coming to use a LAN (Local Area Network) to save the image in the database. We developed a remote control system in which SEM image and Control interface is indicated on the WEB Browser. in this system, SEM can be controlled by an external (client) PC installed in a general WEB Browser(Internet Explorer). Accordingly, operation interface can be indicated on the WEB browser.

A JSM-6700F is connected to a LAN, and so the microscope can be controlled by a client PC. Figure. 1 shows the block diagram of SEM remote control system. The JSM-6700F has two lines to the LAN for image transfer and communication with the SEM control. in order to transfer the image, the image size squeezes from 1280 × 1024-pixels (SEM image size) to 640 × 480-pixels for quick transfer. The image signal (640 × 480-pixels) is connected to the video server only, and then the image transfers to the client PC via LAN . The SEM control communicates with client PC for external command. On the other hand, the SEM control interface and the image are indicated on WEB Browser (Internet explorer) as shown Figure 2. The SEM control interface is composed of the SEM image area and the SEM control part. The SEM image indicates the 640×480-pixels live image. This pixel number was an image quality sufficient to adjust the focus. When a high quality image is necessary, the image of 1280 × 1024-pixels is saved on the the SEM(server) side by choosing the image save button.

The software DigitalMicrograph (DM) by Gatan, Inc., is a popular software platform for digital imaging in microscopy. in a service-oriented microscopy laboratory, a large number of images from many different samples are generated each day. It is critical that each printed image is properly labeled with sample identification and a description before printing. with DM, a script language is provided: from this, various analyses can be designed or customized and repetitive tasks can be automated. This paper presents the procedures and DM scripts needed to perform these tasks. Due to the major software architecture change between version 2.5x and version 3.5x, each will be discussed separately.

DM Version 2.5.8 (on Macintosh®)A “Data Bar” mechanism is provided in this version of DM. Using the “Edit→Data Bar→Define and Add Data Bar...“ menu command specifies data bar items (e.g., scale bar, microscope operator) to be included in the image. in addition, other annotations (text, line, rectangle, and oval) can be included as part of “Data Bar.” This is done by first selecting the desired annotation on the image and then using the “Edit→Data Bar→use AS Default Data Bar...” menu command. After defining data bar items, executing the menu command adds these data bar items to the image.

The annual concrete consumption of two tons for every U.S. citizen makes it the most widely used construction material. Preserving this large concrete infrastructure requires an understanding of the deterioration and failure processes. Concrete exhibits composite behavior at several length scales (millimeter, micron, and submicron). in conjunction with this complex micro structure, damage processes such as crack formation and void collapse occurs at these various length scales. The diverse length scales combined with the large dimensions (meters) of concrete systems necessitates examination of macroscopic areas/volumes at microscopic magnifications to characterize damaged structures. This translates into examining large specimens, capturing a large number of images, and performing analysis on each image. Automating the image capture and image analysis greatly reduces the labor in evaluating damaged concrete structures.

Automated specimen positioning and image capture from an environmental scanning electron microscope (ESEM, ElectroScan 2020) and automated image analysis (Noesis Visilog 5.3 image analysis software) were synthesized with statistical and stereo logical principles into a comprehensive system for quantitative micro structural investigation of concrete.

We have developed a new Electron Probe Microanalyzer (EPMA), JXA-8100/8200, with improved basic capabilities such as X-ray intensities of wavelength dispersive spectrometers (WDS), imaging functions, automated functions and analysis software. Fig. 1 shows the appearance of JXA-8200, WD/ED combined microanalyzer. in this session, we report mainly on the improved imaging functions, automated functions and analysis software.

The JXA-8100/8200 is the first EPMA in the world to feature 1280 x 1024 pixels high resolution live scanning image display. Regarding scanning image, two or four different signal live images, of course including X-ray images, can be displayed simultaneously. Further, image mixing is also possible to display. On the high resolution image, an operator can choose the probe position or the stage position by mouse clicking. The stage position can also be chosen on the optical microscope (OM) live image. Another new “Swing Mouse” function, which is the seamless movement of mouse pointer between the scanning image display and the computer display, has been developed.

Advanced automated functions, such as autofocus, auto astigmatism and auto contrast brightness, are effective to optimize the scanning image.

A typical TEM specimen grid provides approximately a 2×2 mm area that is available for imaging. in order to identify and locate suitable targets on the grid the microscopist must usually inspect the grids at magnifications that allow for only a small area of the grid in the field of view at a time. Systematically searching the grid and mentally keeping track of relative locations presents a challenge for a microscopist especially as the image normally rotates as the magnification is changed. We present an automated technique that creates a very low magnification (VLM) image of the entire available imaging area on the grid. The VLM image can then be used as a reference map for searching the grid at high magnification.

VLM images were obtained at a nominal magnification of 57x using a Philips CM200 TEM equipped with a Gatan CCD camera. The VLM image of the entire 2 mm specimen grid can be created using a mosaic of 49 images, each 512×512 pixels in size. The sampling distance between the images in the 7×7 array is 300μm and the pixel size is 680nm. The images are then automatically tiled and re-sampled to form a final VLM matrix of 4K×4K with a pixel size of 730nm. The resampling factors for the tiling operation include the scale and the relative angle between the camera and goniometer axes. These factors are determined automatically from a calibration process that characterizes the goniometer.

We have been developing a software system, called Leginon, for the control and acquisition of images from a transmission electron microscope. This software allows for the automatic acquisition of images under low dose conditions from a specimen embedded in vitreous ice. Thousands of images are collected during each session at the microscope. These images are collected at various magnifications and under a variety of conditions. For example, images are collected of the entire grid, individual grid squares, individual holes within the square, and several high magnification images are acquired for one to several targets within each hole (figure 1). in addition, for each identified hole an image is acquired at the low dose focus position. Power spectra are also calculated for each of the high magnification images as well as for the focus images. For managing these images we are using a relational database management system. We have also developed a bench of tools to store, access, and display the data as well as tools for extracting information from the database.

A database is a structured collection of data in which the information is classified by categories. Each category is then stored in a table and these tables are linked together by defined relations. Data may be combined from several tables on request. We have been using MySQL as our relational database management system. This allows us both to manage the images and to refer each image to its real context. in other words, through the database, we can keep track of information related to the microscope settings (e.g. defocus, electron dose, magnification, goniometer coordinates etc.), the type of image, and overall information about the specimen and the experiment.

For several years, we have been involved in developing a completely automated system for cryoelectron microscopy, which will allow a 3D electron density map of a macromolecular structure to be generated from a specimen inserted into the microscope with no manual intervention required by an operator. Towards this goal, we have developed a system, called Leginon, to automatically acquire images from the electron microscope. In order to integrate the 3D reconstruction process with the data acquisition step we need then to automatically identify and segment objects from the electron micrographs. This presents a challenge, particularly in the case when micrographs are acquired very close to focus, which results in an image with extremely low contrast. We present here the results of a two-stage automated process for detecting and selecting filamentous structures from images of this kind. Images are acquired in defocus pairs; a near-to-focus (NTF) image is acquired first, followed by a far-from-focus (FFF) image. In the first stage of the process, a threelevel perceptual organization algorithm is used to identify and segment filaments in the FFF image. In the second stage the NTF image is aligned to the FFF image using phase correlation techniques and the filaments are selected at the same coordinates as were identified in the FFF image.

In humans, perceptual organization is the ability to immediately detect structural relationships such as co-linearity, parallelism, and connectivity among image elements. Researchers in human vision perception have long recognized the importance of the common rules by which our visual system attempts to group information. Some of these rules include proximity, similarity, common fate, continuation and closure.

Over the more than twenty years of operation of the 1.2 MeV AEI/Kratos HVEM at Argonne (Fig 1.), the primary emphasis by the user community has been on a wide variety of in situ irradiation effects studies involving both electrons and ions. in many cases it has been essential for the success of experiments for the professional technical staff of the Facility to develop specialty devices and techniques. This contribution deals with one such system which we have developed, namely, the design, assembly and operation of a system for in situ automated measurement of specimen electrical resistivity as a function of temperature in the range 15-135 K. A major problem is associated with this situation, which involves a small gauge-section conductor, requiring stable amplification and measurement of very minute electrical signals. The specific set of experiments for which this application was developed involved determining the effect of electron irradiation on the superconducting transition temperature Tc of YBaCuprate (YBCO) in order to distinguish between two possible theories of the conduction mechanism. in this type of experiment the critical temperature Tc is determined from a plot of electrical resistivity as a function of temperature following irradiation at 100 K at zero applied magnetic field to preclude the Meissner Effect. in these experiments Tc is 89 K prior to electron irradiation and drops to <15 K with increasing electron fluence. The analysis also revealed an oxygen-disorder-induced superconductor to normal-metal transition after sufficient electron fluence. Fig. 2 shows typical measurement results for two temperature ramps. The detailed results of these experiments are described elsewhere by Giapintzakis et al.

Reconstruction of phase images from off-axis electron holograms recorded in a field-emission TEM was for many years a tedious and time-consuming process. The advent of digital imaging systems and ever-faster desktop computers has recently resulted in processing times of a few seconds for a useful phase reconstruction. However, a goal of the “holographer” has been to view the phase images essentially in “live-time”, that is, at least at a few frames per second. This enables effective observation of phase change, through “a window to the phase world,” while the sample is being searched. Chen et al. used a mixture of digital and analog techniques to obtain phase images at nearly TV scan rates. Because optical lens systems provide the fast Fourier processing required for phase reconstructions, their geometry allows phase images to be displayed while the sample is being moved and/or timedependent specimen changes are occurring. Voelkl et al. have described a purely digital system for phase reconstructions from electron holograms that provides several phase images per second, and has several advantages over the mixed system. The refresh rate is presently limited more by the readout time of the slow-scan digital camera than by the computational time of the computer.

Dental enamel is the most durable bioceramics produced by a vertebrate as it is designed to perform masticatory functions throughout its lifetime. The understanding of the mechanism of enamel formation and effects of proteins during the biomineralization process are fundamental issues, essential for both potential enamel regeneration and as a base for synthesis, via self-assembly, of biomimetic composites.

The biomineralization process of enamel is carried out by ameloblast cells that line the inner enamel epithelium and secrete an extracellular protein matrix onto a mineralized dentin surface at the dentin-enamel junction (DEJ). A major matrix protein, amelogenin, is believed to regulate the mineralization of hydroxyapatite (HAP) in the enamel tissue. It has been shown to undergo self-assembly in vitro and in vivo to form nanospheres of ∼20nm in diameter. Previous TEM studies have shown that the nanospheres align along the length (c-axis) of hydroxyapatite (HA) crystals. There are two domains, namely A (residues 1-42) and B (residues 157-173), that control the self-assembly behavior of the nanospheres.

Electron probe microanalysis has been a useful method for biological research for some years. Its advantages include the low background of x-ray spectra and range to include characteristic peaks of most biological elements. The traditional method of analyzing biological X-ray spectra filtering both the experimental and reference spectra with a top-hat filter, before performing a linear least squares fit, to remove the slowly varying background. We show that, as expected, this procedure introduces an additional error in the quantitation results, and reduces the sensitivity and precision of this method. Our experience with quantitating trace elements with EELS shows that direct fit of unfiltered spectra yields the highest sensitivity, when the data contain only statistical errors. Biological specimens usually contain high K and low Ca, and in X-ray spectra the K Kβ peak is overlapped with the Ca Kα peak. A small error in extracting the K signal will dramatically affect the quantitation of Ca. We demonstrate here the application of a direct fitting procedure, which focuses on the Ca and K region of the X-ray spectra (2800 ∼ 4900 eV), and yields smaller fitting uncertainties compared to the traditional filtered fit method in trace Ca quantitation.

The low survival of sugar maple (Acer saccharum) seedlings growing in unglaciated acidic forest soils (pH<4.0) in north-central Pennsylvania may be due in part to aluminum (Al) or manganese (Mn) phytotoxicity. Although foliar analysis has shown high concentrations (>2,000 ppm) of Mn, the location of potentially toxic elements in leaves or absorptive fine roots has not been studied in field-grown seedlings. This study of vascular (xylem and phloem) cells was conducted to determine by energy-dispersive X-ray microanalysis (EDX) whether Mn and/or Al could be detected in fine root or leaf midvein vascular cells. A secondary objective was to determine if vascular cells prepared by chemical fixation (CF) for conventional TEM or by freeze-substitution (FS) for EDX showed similar ultrastructural characteristics.

Four sugar maple seedlings from an unglaciated field site (Potter County PA) were excavated, and roots were washed carefully. Two mm-long segments of fine roots and leaf midveins from each seedling were immersed in 3% glutaraldehyde for 6 hours, transferred to 0.1 M phosphate buffer, and transported to the Delaware Research Lab where tissues were post-fixed in OsO4 (in buffer), dehydrated in ethanol, and embedded in PolyBed-Araldite epoxy resin (CF method).

The airways are lined with a thin film consisting of two layers. A watery layer, the airway surface liquid (ASL) surrounds the cilia and its major function is to serve as a medium for ciliary boating of mucus out of the respiratory system. The other layer is a viscous mucus layer consisting mainly of airway cellular secretions and cellular debris.

The composition and effects of altered concentrations of the constitutive elements in the ASL and mucus layer remain a mystery. in human patients, attempts have been made to determine the composition of these layers by sampling with a filter paper by touching it to the mucosa and wetting it by capillary action. This material was then removed from the filter paper and quantitative analyses using energy dispersive x-ray spectrometry (EDS) and a Hall’s thin film correction routine performed on the residue. Although differences were detected between normal and pathological material, these results did not reflect the composition of the individual layers but an average of both.

Attempts have been made to determine the elemental composition of these layers in intact and in cultured rabbit trachea, bovine trachea and hamster trachea.

To make specimens for the biological materials, especially the retina and palpebral conjunctiva, and to detect the elements and organic substances.

Twelve-week-old male Wistar Kyoto rats were used. Immediately after the eyes were enucleated under pentobarbital anesthesia, the eyeballs and eyelids were put into liquid nitrogen without any fixation and covered by O.C.T. compound (TISSUE-TEK, Miles Inc.). Semi-thin sections (5 μm) were cut with a cryomicrotome at −30 °C (Zeiss, Germany). Sections of the retina and palpebral conjunctiva were placed on siliconwafer plates 10 × 7 × 1 mm and air-dried for 30 min or freeze-dried for 8 hrs. A metal microscope (Leica- DMRBE) was used to confirm the morphological features of the unstained conjunctiva and retina on the siliconwafer plate prior to analysis with a TOF-SIMS microscope. Without coating, the specimens were examined with a TOF-SIMS microscope (TRIFID) (Physical Electronics, USA). Positive and negative ion images were examined with a Ga+ source at an acceleration voltage of 15 kV. Secondary ion acceleration voltage of 4.5 keV was used. The amount of trace elements was analyzed by Wincadence (Physical Electronics, USA) software.

Digital fluorescence microscopy is now a standard tool for determining the localization of cellular components in fixed and living cells. Two fundamentally different imaging technologies are available for imaging fluorescently labelled cells and tissues, in either the fixed or living state. The laser scanning microscope uses a diffraction-limited focused beam to scan the sample and develop an image point by point. in addition, a pinhole placed in a plane confocal to the specimen prevents emitted out-of focus fluorescence from reaching the photomultiplier tube (PMT) detector. By combining spot illumination and selection of infocus fluorescence signal, the laser scanning confocal microscope (LSCM) creates an image of the specimen largely free of out-of-focus blur. By contrast, a wide-field microscope (WFM) illuminates the whole specimen simultaneously and detects the signal with a spatial array of point detectors, usually a charge-coupled device camera (CCD). This approach collects an image of all points of the specimen simultaneously and includes all the out-of-focus blurred light. Subsequent restoration by iterative deconvolution generates an estimate of the specimen, largely free of out-of-focus blur. While many other fluorescence imaging modalities exist, these two methods represent the majority of the fluorescence imaging systems currently in use in biomedical research.

Here we show a confocal microscope system where a vertical-cavity surface-emitting laser (VCSEL) array is introduced for the parallel probing beams.

A VCSEL array is a semiconductor laser device that emits circular beams vertically to the surface of the chip, which can directly be used as confocal laser microscope applications. The VCSEL device itself can be the parallel beam source of the confocal laser system, whereas in conventional Nipkow disc-based system, several lenses are required to form probing beams. Figure 1 shows the basic structure of VCSEL-based confocal microscope systems. Since there is typically no correlation between individual channels of VCSEL, there are no interferences among dense parallel beam arrays. Additionally, in the case of Nipkow disc system, the signal to noise ratio of the detected light is another problem: some of the light from the source laser reflects at the surface of the disc, which eventually could be detected by the sensor as noise. in the proposed system no such problem occurs as shown in Fig. 2(a). in addition, “all” optical beam from VCSEL is used as probing beam whereas most of the power is lost at the disc as compared in Fig. 2(b). in addition, pixel parallel illumination control is also achievable by controlling each channel of the VCSEL so that the dynamic range of the detected signal is maximized.

Continuing improvements on laser scanning microscopy now allow easy acquisition of multi-gigabyte-sized data sets from single living specimens. These multi-dimensional data comprise stacks of serial optical sections (3D) collected repeatedly over time (4D), often recording separate signal channels simultaneously (5D). in our work imaging developmental and cell biological processes in living embryos, we try to take advantage of the full scope of spatial and temporal detail that microscopes can now accumulate. Aside from the obvious difficulties of data management and archival, we still struggle to visually appreciate both the holistic view of the specimen and the smaller dynamic details that we know are captured within these recordings. Ideally, the multi-dimensional microscopist should view and analyze the specimen as the full set of tens of thousands of images. Although strides have been made, visualization software and computer hardware for browsing and analyzing multi-dimensional data are not yet up to the task of giving the microscopist full kinesthetic access to the thriving subject that the microscope hardware and software have captured. Several pieces of software allow the viewer to fluidly browse the original optical sections through space and over time. Moving beyond the constraints of the raw image plane by rendering, re-slicing, digitally dissecting, and animating the time-varying volumes is the task that is currently so demanding that all approaches are only partially satisfactory.