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[[File:SingleParticleAnalysis.png|thumb|right|Single particle analysis segments and averages many particles from a sample, allowing for computer algorithms to process the individual images into a combined "representative" image. This allows for improvements in signal to noise, and can be combined with [[deconvolution]] to provide limited improvements to spatial resolution in the image.]]
'''Single particle analysis''' is a group of related computerized image processing techniques used to analyze images from [[Transmission electron microscope|transmission electron microscopy]] (TEM).<ref name="Frank">{{Cite book|first=Joachim |last=Frank |title=Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state |publisher=Oxford University Press |___location=Oxford |year=2006 |pages= |isbn=978-0-19-518218-7 |url=https://books.google.com/?id=vWaSRUjicbgC}}{{Page needed|date=August 2010}}</ref> 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 [[virus]]es. Individual images of stained or unstained particles are very [[Signal noise|noisy]], and so hard to interpret. 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 [[Transmission electron microscopy#Three dimensional imaging|three-dimensional reconstruction]] of the particle. Using [[cryogenic transmission electron microscopy|cryo-electron microscopy]] it has become possible to generate reconstructions with sub-nanometer [[Resolution (electron density)|resolution]] and near-atomic resolution
==Techniques==
Single particle analysis can be done on both [[negative stain|negatively stained]] and vitreous ice-embedded [[Cryogenic transmission electron microscopy|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), in the past, were collected on film are digitized using high-quality scanners or using built-in [[charge-coupled device|CCD]] detectors coupled to a phosphorescent layer. Now it is common to use direct electron detectors to collect images. The image processing is carried out using specialized software [[Software tools for molecular microscopy|programs]] (for instance
===Alignment and classification===
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===Tilt methods===
The specimen stage of the microscope can be tilted (typically along a single axis), allowing the single particle technique known as random conical tilt.<ref name="RCT">{{Cite journal|vauthors=Radermacher M, Wagenknecht T, Verschoor A, Frank J |title=Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli |journal=Journal of Microscopy |volume=146 |issue=Pt 2 |pages=113–36 |date=May 1987 |pmid=3302267 |doi=10.1111/j.1365-2818.1987.tb01333.x}}</ref> An area of the specimen is imaged at both zero and at high angle (~60-70 degrees) tilts, or in the case of the related method of orthogonal tilt reconstruction, +45 and
3D reconstructions from random conical tilt suffer from missing information resulting from a restricted range of orientations. Known as the missing cone (due to the shape in reciprocal space), this causes distortions in the 3D maps. However, the missing cone problem can often be overcome by combining several tilt reconstructions. Tilt methods are best suited to negatively stained samples, and can be used for particles that adsorb to the carbon support film in preferred orientations. The phenomenon known as charging or beam-induced movement makes collecting high-tilt images of samples in vitreous ice challenging.
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