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DOI: 10.22184/1993-8578.2020.13.6.340.344
Scanning probe microscopy provides three-dimensional visualization of viruses in various media, for example, liquid and air. In addition to three-dimensional topography, it is possible to measure such properties as mechanical rigidity, adhesion, crystallization tendency, aggregation of particles on a substrate, etc. Characterization of animal and human viruses is the subject of numerous studies due to their potential harm to higher organisms.
Scanning probe microscopy provides three-dimensional visualization of viruses in various media, for example, liquid and air. In addition to three-dimensional topography, it is possible to measure such properties as mechanical rigidity, adhesion, crystallization tendency, aggregation of particles on a substrate, etc. Characterization of animal and human viruses is the subject of numerous studies due to their potential harm to higher organisms.
Теги: 3-d topography 3d-топография scanning probe microscopy virology вирусология сканирующая зондовая микроскопия
The scanning probe microscopy techniques were applied to imaging viruses in plants, animals and humans [1] shortly after the invention of scanning tunneling microscopy in 1982 [2, 3] and atomic force microscopy [4] in 1986. From the very first attempts to visualize viruses, it became obvious that a new highly informative tool appeared that can be used in basic and practical sciences, including virology and medicine. Until now, certain unique information can only be obtained using the scanning probe microscopy methods. This information includes experimental data on the mechanical properties of the virus (mechanical rigidity and stability), adhesive properties, and binding strength.
It is worth noting that other powerful methods based on transmission electron microscopy (TEM) should not be viewed as competing with scanning probe microscopy in the environmental studies. Transmission microscopy techniques provide the images of viral particles in a vacuum, which perfectly complements the data obtained with probe microscopy.
A feature of the probe microscopy as distinct from other methods is sample preparation. It does not require any special skills. Three types of substrates are used in probe microscopy: mica, graphite, and glass. Mica is hydrophilic, making it easier to coat samples with the virus. Freshly split graphite is hydrophobic. For both graphite and mica, the top layer can be removed to create an ideal substrate for probe microscopy. For graphite of the highest quality, the distance between the terraces can be up to 10 microns. Glass is used much less frequently and is not as uniformly flat as graphite and mica. Glass usually has a wavy relief with nanometer-sized amplitude. The observed glass topography can make it difficult to accurately measure the virus size.
Adhesion of mica and graphite can be illustrated by examining the tobacco mosaic virus. Depending on the substrate, the same concentration of particles in the sample can differ for graphite and mica [5].
Functionalized substrates are used in sensory applications. In this case, the substrates can be coated with antibodies [6], aptamers [7] and synthetic receptors [8, 9].
When preparing a sample with viral particles, it is desirable to exclude the ingress of foreign objects, which especially impede visualization of settled particles from the buffer. Distilled water can be used to dilute the sample: after applying the suspension to the substrate and prolongation phase, the sample can be washed. The prolongation duration depends on the sample and can range from a few seconds to 30 minutes.
During scanning, the actual sizes of the virus particles may differ. Sometimes, due to interaction of the virus with the substrate, the height may be less than the real one. Also, the particle height depends on the substrate: the virus can be higher on graphite than on mica (the difference in the observed heights of the tobacco mosaic virus is about 1 nm). Due to the shape of the cantilever tip, the particles may appear wider than they actually are. This is the so-called cantilever broadening effect [10]. The exact diameter of the virus can be observed in the crystalline form [11].
SOFTWARE FOR PROCESSING THE OBTAINED RESULTS
After scanning, you need to process the results. Image processing is another highly artistic task. Interpretation of the results is highly dependent on how the image has been processed.
Most software packages have a built-in geometric measurement and transformation service, which includes [12]:
construction of cross-sections and histograms,
measurement of distances, angles, lengths of broken lines,
determination of surface roughness,
measurement of the perimeter of the selected area,
automatic determination of the length of the isoline, and its limited area and surface volume,
removal of the middle slope,
construction of three-dimensional images,
selection of the most suitable color palette.
Recognition of the same shape objects is one of the important tasks in probe microscopy. The human eye quickly finds a particular shape of interest in an image. However, clear mathematical criteria are needed to automate this process. When analyzing a sample with a smooth surface, the image has a uniform background against which automatic search is easily accomplished using mathematical algorithms built into most image processing programs for optical, electron and probe microscopy.
FemtoScan Online is a user-friendly image processing software that accepts over 100 different data formats developed by the existing and now extinct companies which microscopes are used in laboratories around the world. The program is convenient for analyzing large images, since two images can be displayed simultaneously: an overview image with a "sliding" area and a detailed image of a "sliding" area. When you move the selected area, the detail image, the sectional area and the Fourier image change synchronously. This function is used when searching for objects of interest. Also in FemtoScan Online, 3D images can be viewed on a stereo monitor. The program carries out prepress preparation and preparation of presentations in a convenient and simple way: lighting, setting colors and fonts, creating 3D images and video with flying over the surface, stereo mode for 3D images [13, 14].
Despite a visible progress in visualization of viruses using probe microscopy, there are still many unresolved problems. Currently, the data of probe microscopy of viruses is not systematized, a descriptive atlas of images and morphology detected using high-resolution microscopy has not been created. This is one of the tasks of the probe microscopy laboratory of the physics and chemistry faculties of the Lomonosov Moscow State University. ■
It is worth noting that other powerful methods based on transmission electron microscopy (TEM) should not be viewed as competing with scanning probe microscopy in the environmental studies. Transmission microscopy techniques provide the images of viral particles in a vacuum, which perfectly complements the data obtained with probe microscopy.
A feature of the probe microscopy as distinct from other methods is sample preparation. It does not require any special skills. Three types of substrates are used in probe microscopy: mica, graphite, and glass. Mica is hydrophilic, making it easier to coat samples with the virus. Freshly split graphite is hydrophobic. For both graphite and mica, the top layer can be removed to create an ideal substrate for probe microscopy. For graphite of the highest quality, the distance between the terraces can be up to 10 microns. Glass is used much less frequently and is not as uniformly flat as graphite and mica. Glass usually has a wavy relief with nanometer-sized amplitude. The observed glass topography can make it difficult to accurately measure the virus size.
Adhesion of mica and graphite can be illustrated by examining the tobacco mosaic virus. Depending on the substrate, the same concentration of particles in the sample can differ for graphite and mica [5].
Functionalized substrates are used in sensory applications. In this case, the substrates can be coated with antibodies [6], aptamers [7] and synthetic receptors [8, 9].
When preparing a sample with viral particles, it is desirable to exclude the ingress of foreign objects, which especially impede visualization of settled particles from the buffer. Distilled water can be used to dilute the sample: after applying the suspension to the substrate and prolongation phase, the sample can be washed. The prolongation duration depends on the sample and can range from a few seconds to 30 minutes.
During scanning, the actual sizes of the virus particles may differ. Sometimes, due to interaction of the virus with the substrate, the height may be less than the real one. Also, the particle height depends on the substrate: the virus can be higher on graphite than on mica (the difference in the observed heights of the tobacco mosaic virus is about 1 nm). Due to the shape of the cantilever tip, the particles may appear wider than they actually are. This is the so-called cantilever broadening effect [10]. The exact diameter of the virus can be observed in the crystalline form [11].
SOFTWARE FOR PROCESSING THE OBTAINED RESULTS
After scanning, you need to process the results. Image processing is another highly artistic task. Interpretation of the results is highly dependent on how the image has been processed.
Most software packages have a built-in geometric measurement and transformation service, which includes [12]:
construction of cross-sections and histograms,
measurement of distances, angles, lengths of broken lines,
determination of surface roughness,
measurement of the perimeter of the selected area,
automatic determination of the length of the isoline, and its limited area and surface volume,
removal of the middle slope,
construction of three-dimensional images,
selection of the most suitable color palette.
Recognition of the same shape objects is one of the important tasks in probe microscopy. The human eye quickly finds a particular shape of interest in an image. However, clear mathematical criteria are needed to automate this process. When analyzing a sample with a smooth surface, the image has a uniform background against which automatic search is easily accomplished using mathematical algorithms built into most image processing programs for optical, electron and probe microscopy.
FemtoScan Online is a user-friendly image processing software that accepts over 100 different data formats developed by the existing and now extinct companies which microscopes are used in laboratories around the world. The program is convenient for analyzing large images, since two images can be displayed simultaneously: an overview image with a "sliding" area and a detailed image of a "sliding" area. When you move the selected area, the detail image, the sectional area and the Fourier image change synchronously. This function is used when searching for objects of interest. Also in FemtoScan Online, 3D images can be viewed on a stereo monitor. The program carries out prepress preparation and preparation of presentations in a convenient and simple way: lighting, setting colors and fonts, creating 3D images and video with flying over the surface, stereo mode for 3D images [13, 14].
Despite a visible progress in visualization of viruses using probe microscopy, there are still many unresolved problems. Currently, the data of probe microscopy of viruses is not systematized, a descriptive atlas of images and morphology detected using high-resolution microscopy has not been created. This is one of the tasks of the probe microscopy laboratory of the physics and chemistry faculties of the Lomonosov Moscow State University. ■
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