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Single particle analysis
Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses. Individual images of stained or unstained particles are very noisy, making interpretation difficult. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with sub-nanometer resolution and near-atomic resolution first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well. Single particle analysis can also be performed by inductively coupled plasma mass spectrometry (ICP-MS).[relevant?]
Single particle analysis can be done on both negatively stained and vitreous ice-embedded transmission electron cryomicroscopy (CryoTEM) samples. Single particle analysis methods are, in general, reliant on the sample being homogeneous, although techniques for dealing with conformational heterogeneity are being developed.
Images (micrographs) are taken with an electron microscope using charged-coupled device (CCD) detectors coupled to a phosphorescent layer (in the past, they were instead collected on film and digitized using high-quality scanners). The image processing is carried out using specialized software programs, often run on multi-processor computer clusters. Depending on the sample or the desired results, various steps of two- or three-dimensional processing can be done.
In addition, single particle analysis can also be performed in an individual particle mode using an ICP-MS unit.
Biological samples, and especially samples embedded in thin vitreous ice, are highly radiation sensitive, thus only low electron doses can be used to image the sample. This low dose, as well as variations in the metal stain used (if used) means images have high noise relative to the signal given by the particle being observed. By aligning several similar images to each other so they are in register and then averaging them, an image with higher signal-to-noise ratio can be obtained. As the noise is mostly randomly distributed and the underlying image features constant, by averaging the intensity of each pixel over several images only the constant features are reinforced. Typically, the optimal alignment (a translation and an in-plane rotation) to map one image onto another is calculated by cross-correlation.
However, a micrograph often contains particles in multiple different orientations and/or conformations, and so to get more representative image averages, a method is required to group similar particle images together into multiple sets. This is normally carried out using one of several data analysis and image classification algorithms, such as multi-variate statistical analysis and hierarchical ascendant classification, or k-means clustering.[citation needed]
Often data sets of tens of thousands of particle images are used, and to reach an optimal solution an iterative procedure of alignment and classification is used, whereby strong image averages produced by classification are used as reference images for a subsequent alignment of the whole data set.
Image filtering (band-pass filtering) is often used to reduce the influence of high and/or low spatial frequency information in the images, which can affect the results of the alignment and classification procedures. This is particularly useful in negative stain images. The algorithms make use of fast Fourier transforms (FFT), often employing Gaussian shaped soft-edged masks in reciprocal space to suppress certain frequency ranges. High-pass filters remove low spatial frequencies (such as ramp or gradient effects), leaving the higher frequencies intact. Low-pass filters remove high spatial frequency features and have a blurring effect on fine details.
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Single particle analysis AI simulator
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Single particle analysis
Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses. Individual images of stained or unstained particles are very noisy, making interpretation difficult. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with sub-nanometer resolution and near-atomic resolution first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well. Single particle analysis can also be performed by inductively coupled plasma mass spectrometry (ICP-MS).[relevant?]
Single particle analysis can be done on both negatively stained and vitreous ice-embedded transmission electron cryomicroscopy (CryoTEM) samples. Single particle analysis methods are, in general, reliant on the sample being homogeneous, although techniques for dealing with conformational heterogeneity are being developed.
Images (micrographs) are taken with an electron microscope using charged-coupled device (CCD) detectors coupled to a phosphorescent layer (in the past, they were instead collected on film and digitized using high-quality scanners). The image processing is carried out using specialized software programs, often run on multi-processor computer clusters. Depending on the sample or the desired results, various steps of two- or three-dimensional processing can be done.
In addition, single particle analysis can also be performed in an individual particle mode using an ICP-MS unit.
Biological samples, and especially samples embedded in thin vitreous ice, are highly radiation sensitive, thus only low electron doses can be used to image the sample. This low dose, as well as variations in the metal stain used (if used) means images have high noise relative to the signal given by the particle being observed. By aligning several similar images to each other so they are in register and then averaging them, an image with higher signal-to-noise ratio can be obtained. As the noise is mostly randomly distributed and the underlying image features constant, by averaging the intensity of each pixel over several images only the constant features are reinforced. Typically, the optimal alignment (a translation and an in-plane rotation) to map one image onto another is calculated by cross-correlation.
However, a micrograph often contains particles in multiple different orientations and/or conformations, and so to get more representative image averages, a method is required to group similar particle images together into multiple sets. This is normally carried out using one of several data analysis and image classification algorithms, such as multi-variate statistical analysis and hierarchical ascendant classification, or k-means clustering.[citation needed]
Often data sets of tens of thousands of particle images are used, and to reach an optimal solution an iterative procedure of alignment and classification is used, whereby strong image averages produced by classification are used as reference images for a subsequent alignment of the whole data set.
Image filtering (band-pass filtering) is often used to reduce the influence of high and/or low spatial frequency information in the images, which can affect the results of the alignment and classification procedures. This is particularly useful in negative stain images. The algorithms make use of fast Fourier transforms (FFT), often employing Gaussian shaped soft-edged masks in reciprocal space to suppress certain frequency ranges. High-pass filters remove low spatial frequencies (such as ramp or gradient effects), leaving the higher frequencies intact. Low-pass filters remove high spatial frequency features and have a blurring effect on fine details.