Low Voltage Scanning Electron Microscopy

The scanning electron microscope is a significant tool which is used in laboratory analysis. Moreover, it has not been fully utilized in its applications because of its size. Advanced technological developments in the recent time, has enabled the emergence of low-voltage micro-columns as part of miniature scanning electron microscope. This in turn has developed an attractive feature in low-voltage operations making it a powerful analytical tool, which can perform a variety of applications.

Low voltage scanning electron microscopy (LVSEM) is part of the SEM application which utilizes beam energies which are below five keV. This tool does not have a high electron beam range. It produces less three dimensional images in contrast to the high resolution scanning electron microscopy, but which contains substantial detailed information. Moreover, concerns of using the tool in low energy, that stops the power of electrons, can cause enhanced radiation damage in the analysis of a sample. Despite this aspect, damage as a result of small electron on range, are confined to the near thin region or surface of the specimen.

Advances in the probable electron optics and electron sources, still promise improved performances for the LVSEM. These improvements in electron optics have made it possible for the electron energy, to cover a wider range without significant degradation towards imaging quality of the low voltage scanning electron microscope. When using low voltages, surface sensitivity of a given sample is enhanced with secondary electron emission. The resolution of the low voltage SEM is tested through analysis of various criteria combinations, which entail resolved gaps and gray levels spotted in the image. Distortion of the image is reduced by ensuring contrast and maximizing visibility of edges in low voltages. Finer details are obtained with evidence of gray levels at a good range.

The aim of this paper is to demonstrate the usage of LVSEM in various applications in research and commercial applications.

The Low Voltage Scanning Electron Microscopy
The low voltage scanning microscope can be described as an electron microscope that operates with the acceleration of voltages in few kiloelectronvolts (keV). Although, the use of low voltage electron microscopy application and techniques cannot be replaced with the conventional high powered electron microscopes, the tool is significantly being appreciated in various disciplines and fields. LVSEM has significant advantages concerning imaging, where it allows high quality images to be analyzed for various samples, which would have otherwise been impossible to visualize using other microscopy techniques.

At the time, there is just one commercially available LVSEM, which has few key changes for it to operate under the 5 keV electron source in TEM mode analysis. In addition, the instrumentation of advanced microscopy has in the recent years grown enormously with the consequent development of LVSEM. The instrument allows imaging in high resolution of samples in delicate biological structures. Minimal requirements in coating produce clear images at high resolution, which had only been attainable through the TEM mode, transmission electron microscopy.

Apart from imaging fine details on the biological materials, LVSEM also integrates isolated cell structures imaging giving impressive details. Three dimensional (3D) images can be attained in the identification and study of macromolecular complexes without structural overlaps. This technique is useful more so towards analyzing molecular components in low quantities, that would be difficult when using negative staining or conventional TEM. The emergence and development of low voltage scanning electron microscopy have evolved from valuable research tools from the past to the present time, while new advances are observed in research and development. The recent LVSEM applications have aided remarkable advances in both research and commercial utilization. Low voltage scanning electron microscopy in research, has become a well founded technique over time, with major contributions in the process of specimen interaction, and consequent modification. New methods in specimen preparation, sample coating and critical point drying, have had a breakthrough in LVSEM development.

Possible formation of low voltage intense beams coupled with a smaller diameter has been a success through developments of sources in field-emissions. Low accelerating voltage has allowed efficient scanning of samples in biology that details impressive results in reduced radiation damage, good contrast, and decreased specimen charging.  Moreover, the technology has improved the aspect of imaging gold metal particles in a given sample at low contrasts, in addition of analyzing flat sample images with minimal coating. Improved sample preparation has been an advantage in the use of LVSEM further adding to the scope of applications coupled with imaging details, which consequently reduce artifacts being liked to conventional preparation techniques, and eliminates the chemical fixation need.  Imaging details in LVSEM entail ultra-rapid freeze-drying and freezing.

The specific biological applications for the LVSEM analytical tool are numerous, and have benefited diverse fields through research and development. Use of this tool has demonstrated the diversity and versatility of LVSEM applications contributing to specific expertise in different respective fields. For the production of high quality results, specimen preparation is a critical aspect with the use of LVSEM imaging. This application demonstrates data acquisition and unique capabilities, with the use of LVSEM applied in different research areas giving detailed results, which can sequentially used to compile substantive analysis (Whitaker, 2003 pg 249). The application of this technique in biology, leads data generation of cellular structures which may include mitotic spindles, nuclear pore complexes, microfilament network observed in bone cells, actomyosin muscle organization in insects among other aspects.

Utilization of low voltage scanning electron microscopy relies in the visualization of de-embedded resin extracted sections of biological materials, in order to provide a unique approach in the ability to examine interior of tissues and cells in incredible details. The process of using LVSEM embeds tissues just like using TEM, and pick out the semi-thick sections of prepared specimen, picked with a copper wire, and transferred to cover glass chips (Torok, 2003 pg 395). Resin that is extracted is then prepared for SEM, coated and critical point dried for analysis. At this point, the specimen can be observed and critically analyzed to produce results for the specific study.

Summary
Use of LVSEM has demonstrated the attractiveness in the capabilities of the low-voltage scanning electron microscopes. The tool has fitted the candidature for a miniature instrument that is capable of fine elemental identification and spatial imaging. Additionally, the small column size observed in the low-voltage SEM, has given it an advantage in the capability of imaging mars-like, and other non-conductive specimens , without requiring complex sample altering preparation. The low-voltage SEM gives clear and high resolution imaging with minimal charging artifacts.

A beam at 2.5kV voltage can readily identify important elemental constituents in a sample level, above a single-percent and quantified with a modest beam and short integration times, with the current used. The precision of low voltage scanning microscopy is sufficient in identifying and resolving spectral results, within substances and samples of interest across various fields of professionalism.

LSEM continues to be a significant and powerful analytical tool, for various applications in the laboratory, more so with the advantages of less critical conductivity of a sample, and the fact that more information is gathered on the surface with minimal penetration of primary electrons into a specimen. Its popularity as an analytical tool, owes to its straightforward data interpretation and operation, resolving surface details with the possibility of accessing various types of information.
Since its inception, the low voltage scanning electron microscope has evolved to encompass a wide range of uses, from a simple instrument with limited applications. Today, the LVSEM is universally applied as a multiuse instrument for commercial and research purposes, which entail chemistry, physics, biology, crystallography, and advanced technologies.

This has been attributed to its versatility in analysis and examination of sample structures surface topography, composition and crystallography, both in heterogeneous inorganic and organic materials. Recent developments in the instrument has enabled it to utilize variable-pressure and also allowing observation of direct chemical reactions in situ. This aspect has enabled the emergence of a new class of analysis in dynamic experiments.

Conclusion
The future directions for LVSEM will require more advances and improvements in order to cater to a broader field of experts and applications. Moreover, this technology has allowed for the delicate imaging of delicate biological materials with minimum requirements in coating. Consequently, it has developed the aspect of generating a wealth of substantial data, which could have been not obtained through other microscopy methods which are currently available.

Low voltage scanning electron microscopy has also enabled detailed study with a range of beam penetration within a sample. There is potential development for the instrument in the future with more improvements towards instrumentation, in the versatility demonstrated by the tool. Spatial resolution and high surface sensitivity have been achieved at low voltage, through also proper sample management.  There are still many unexplored application opportunities with the use of low energy electron beam for analysis, which can provide exciting breakthroughs to complementary and unprecedented information using the low voltage electron microscope.

Continuous improvement will be necessary to increase imaging and resolution of native structures in cell biology analysis, without or less chemical fixation. The LVSEM tool has been a major advantage compared to conventional high voltage microscopy utilities which are consequently better, but has enabled the visualization of finer details in low voltage, and also in three dimensional (3D) spheres.
LVSEM can be integrated with complex applications such as tools within SEMs, in the analysis and dissection of biological materials. Micromanipulation of cells, freeze-drying in SEM, and further applications, will direct LVSEM progress in gradual development and improvement. This will undoubtedly increase the capabilities of LVSEM technology, and further enhance its appreciation to a key research tool in microscopy.

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