Practical method for noise removal in scanning electron microscopy

A new smoothing filter has been developed for noise removal of scanning electron microscopy (SEM) images. We call this the complex hysteresis smoothing (CHS) filter. It is much easier to use for SEM operators than any other conventional smoothing filter, and it rarely produces processing artifacts because it does not utilize a definite mask (which usually has processing parameters of size, shape, weight, and the number of iterations) like a common averaging filter or a complicated filter shape in the Fourier domain. Its criterion for distinguishing noise depends simply on the amplitude of the SEM signal. When applied to several images with different characteristics, it is shown that the present method has a high performance with some original advantages.     

Three-dimensional electron microscopy of entire cells

The digital processing of serial electron-microscope sections containing laser-induced topographical references allows a three-dimensional (3-D) reconstruction of entire cells at a depth resolution of 40–60 nm by the use of novel image analysis methods. The images are directly processed by a video-camera placed under the electron microscope in TEM mode or by the electron counting device in STEM mode. The deformations associated with the cutting of embedded cells are back-calculated by new computer algorithms developed for image analysis and treatment. They correct the artefacts caused by serial sectioning and automatically reconstruct the third dimension of the cells. Used in such a way, our data provide definitive information on the 3-D architecture of cells. This computer-assisted 3-D analysis represents a new tool for the documentation and analysis of cell ultrastructure and for morphometric studies. Furthermore, it is now possible for the observer to view the contents of the reconstructed tissue volume in a variety of different ways using computer-aided display techniques.     

Assessment and correction of geometric distortions in low-magnification scanning electron microscopy images

A method is introduced to assess and correct the geometric distortions which frequently occur in low-magnification scanning electron microscopy (SEM) images. Such images typically exhibit a complex pattern of varying deviations from orthogonality which cannot be adequately corrected by simple geometric transformations such as shifting, scaling, rotation, or shearing. A suitable approach to rectify low-magnification SEM images is polynomial warping, a correction procedure which also accomplishes rubber sheet transformation. To demonstrate the approach, a reference grid for low magnifications has been scanned at 40- and 55-fold magnifications by means of a microanalyzer. Calculated geometric distortions range from 1.5 to 3.5% of the image dimensions; applying polynomial warping, distortions could be reduced to approximately 0.1% of the image dimensions. Because of its easy application and the widespread availability in image processing packages, polynomial warping can be recommended as a routine procedure for rectifying low-magnification SEM images.     

Digital imaging for scanning electron microscopy

The development and application of digital imaging technology has been one of the major advancements in scanning electron microscopy (SEM) during the past several years. This digital revolution has been brought about by significant progress in semiconductor technology, notably the availability of less expensive, high-density memory chips and the development of inexpensive, high-speed, analog-to-digital and digital-to-analog converters, mass storage, and high-performance central processing units. This paper reviews a number of the advantages presented by digital imaging as applied to the SEM and describes a system developed at the National Institute of Standards and Technology for this purpose.     

New generation scanning electron microscopy technology based on the concept of active image processing

A new generation of scanning electron microscopy (SEM) technology is proposed based on the concept of “active image processing.” In order to collect sufficient data for a purpose which is defined in the utilization of active image processing, we may need more devices from among a variety of useful hardware, for example, a digital scan generator with meaningful parameters and an analog-to-digital converter for ultrahigh density recording. After the data acquisition, the application of some digital image processing techniques is certainly effective, because the method in question is specially designed so that the property of obtained data will be suitable for the application of these techniques. The present technology should produce a variety of attractive options in the field of SEM.     

Imperative role of electron microscopy in toxicity assessment: A review

Electron microscope (EM) was developed in 1931 and since then microscopical examination of both the biological and non-biological samples has been revolutionized. Modifications in electron microscopy techniques, such as scanning EM and transmission EM, have widened their applicability in the various sectors such as understanding of drug toxicity, development of mechanism, criminal site investigation, and characterization of the nano-molecule. The present review summarizes its role in important aspects such as toxicity assessment and disease diagnosis in special reference to SARS-COV2. In the biological system, EM studies have elucidated the impact of toxicants at the ultra-structural level in various tissue in conformity to physiological alterations. Thus, EM can be concluded as an important tool in toxicity assessment and disease prognosis.     

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 × 10 × 5 mm³) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 °C, are then fixed at 4 °C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 °C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.     

Reduction in acquisition time of scanning electron microscopy image using complex hysteresis smoothing

Complex hysteresis smoothing (CHS), which was developed for noise removal of scanning electron microscopy (SEM) images some years ago, is utilized in acquisition of an SEM image. When using CHS together, recording time can be reduced without problems by about one-third under the condition of SEM signal with a comparatively high signal-to-noise ratio (SNR). We do not recognize artificiality in a CHS-filtered image, because it has some advantages, that is, no degradation of resolution, only one easily chosen processing parameter (this parameter can be fixed and used in this study), and no processing artifacts. This originates in the fact that its criterion for distinguishing noise depends simply on the amplitude of the SEM signal. The automation of reduction in acquisition time is not difficult, because CHS successfully works for almost all varieties of SEM images with a fairly high SNR.     

Gloss phenomena and image analysis of atomic force microscopy in molecular and cell biology

Proper sample preparation, scan setup, data collection and image analysis are key factors in successful atomic force microscopy (AFM), which can avoid gloss phenomena effectively from unreasonable manipulations or instrumental defaults. Fresh cleaved mica and newly treated glass cover were checked first as the substrates for all of the sample preparation for AFM. Then, crystals contamination from buffer was studied separately or combined with several biologic samples, and the influence of scanner, scan mode and cantilever to data collection was also discussed intensively using molecular and cellular samples. At last, images treatment and analysis with off‐line software had been focused on standard and biologic samples, and artificial glosses were highly considered for their high probability. SCANNING 31: 49–58, 2009. © 2009 Wiley Periodicals, Inc.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

Field-emission scanning electron microscopy of the internal cellular organization of fungi

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.     

Low-temperature scanning electron microscopy in biology

A review of low-temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high-resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient-temperature SEM. This is because frozen-hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen-hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high-resolution LTSEM. The advantages, problems and requirements for this approach are defined.     

An improved scanning method based on characteristics of the human visual system for scanning electron microscopy

An improved scanning method for the scanning electron microscope (SEM) is proposed. Here, quincuncial scanning (sampling) instead of a conventional (raster) scanning is used. This scanning method is very effective for quality improvement of an SEM image obtained under undersampling conditions (rough sampling). The present study focuses on characteristics of the human visual system, specifically the low response of eyes in diagonal directions. When using this method coupled with a high-precision interpolation, the number of pixels necessarily doubles. It is not surprising that it is advantageous for printing. A more important advantage is the fact that SEM images can be acquired with a shorter recording time. Hence, this type of scanning will be helpful for quick and frequent recordings in a "snapshot" mode, which up to now has not been achieved successfully by SEM.     

Virtual reflected-light microscopy

Research on better methods to digitally represent microscopic specimens has increased over recent decades. Opaque specimens, such as microfossils and metallurgic specimens, are often viewed using reflected light microscopy. Existing 3D surface estimation techniques for reflected light microscopy do not model reflectance, restricting the representation to only one illumination condition and making them an imperfect recreation of the experience of using an actual microscope. This paper introduces a virtual reflected-light microscopy (VRLM) system that estimates both shape and reflectance from a set of specimen images. When coupled with anaglyph creation, the system can depict both depth information and illumination cues under any desired lighting configuration. Digital representations are compact and easily viewed in an online setting. A prototype used to construct VRLM representations is comprised only of a microscope, a digital camera, a motorized stage and software. Such a system automatically acquires VRLM representations of large batches of specimens. VRLM representations are then disseminated in an interactive online environment, which allows users to change the virtual light source direction and type. Experiments demonstrate high quality VRLM representations of 500 microfossils.     

Metrics for focusing in extremely noisy scanning electron microscopy condition

Finding a best focused image in very noisy condition is an extremely difficult task for the SEM user. If a performance, which is much higher than that of an expert in focusing, can be achieved in a computer-controlled scanning electron microscope (SEM), it will be very helpful for our field due to the many possible applications. To accomplish this work, we employ a powerful metric-the covariance obtained by a special scanning method. It can select the best focused image from a series of SEM images acquired by altering the focus of the objective lens under an extremely noisy SEM image condition. The noise immunity of the present method is quantitatively evaluated, and it is further improved based on the obtained evaluation result.     

A method for personal expertise-independent evaluation of image resolution in scanning electron microscopy

The two conventional methods currently employed for the evaluation of image resolution in scanning electron microscopy are the gap method and a fast Fourier transform (FFT) method. These can be highly dependent on personal expertise on the distinction between signal information and noise contained in a micrograph. Hence, the present paper proposes an alternative method (referred to as a contrast-to-gradient (CG) method) that can determine the image resolution of a micrograph without requiring personal expertise on the judgment of noise. The image resolution in the CG method is defined as a weighted harmonic mean of the local resolution, which is proportional to the quotient of the threshold contrast divided by the local gradient. The local gradient is calculated from the quadratic function that best fits the local pixel intensities over 5 x 5 pixels. It has been shown that the CG method, compared with the FFT method, has a broader range of applications for various types of images, such as low-contrast, noise-containing, filter-processed, highly directional, and quasi-periodic feature images.     

Representation of three-dimensionally reconstructed objects in electron microscopy by surfaces of equal density

A surface representation algorithm has been developed to visualize the results of three-dimensional reconstructions from projections in electron microscopy. It produces an easily recognizable image of the reconstructed particle and facilitates comprehension of the three-dimensional structure. Thus building of physical models can be avoided in many cases. If such a model is needed the algorithm can be used to establish the correct particle boundary in advance. Some further effort has been made to represent long fibres and visualize inner structures of objects, which are obscured by outer high-density regions. Shading may be used to enhance the depth perception from single views.     

浅析数码显微镜分辨率的影响因素

从奈奎斯特采样定理、光源两方面分析对数码显微图像分辨率的影响,表明在物镜确定的情况下,需要选择合适的摄像头、摄像接头、光源、滤色片等,以满足采样定理,有效发挥物镜的分辨率,准确重建出数字图像。