Single particle analysis: Difference between revisions

Revision as of 15:56, 7 November 2023

Revision as of 12:41, 22 November 2023

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==Techniques==

Single particle analysis can be done on both [[negative stain|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 [https://pubmedconformational heterogeneity<ref>{{cite journal |last1=Lyle |first1=N |last2=Das |first2=RK |last3=Pappu |first3=RV |title=A quantitative measure for protein conformational heterogeneity.ncbi |journal=The Journal of chemical physics |date=28 September 2013 |volume=139 |issue=12 |pages=121907 |doi=10.nlm.nih.gov1063/1.4812791 |pmid=24089719}}</ conformational heterogeneity]ref> are being developed.

Images (micrographs) are taken with an electron microscope using [[Charge-coupled device|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 [[Software tools for molecular microscopy|programs]], often run on multi-processor [[Computer cluster|computer clusters]]. Depending on the sample or the desired results, various steps of two- or three-dimensional processing can be done.

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===Alignment and classification===

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)<ref>{{cite web |last1=Pandithage |first1=Ruwin |title=Brief Introduction to Contrasting for EM Sample Preparation |url=https://www.leica-microsystems.com/science-lab/brief-introduction-to-contrasting-for-em-sample-preparation/ metal|language=en stain]|date=2 usedOctober (if used)2013}}</ref> 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 (geometry)|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 [[Multivariate statistics|multi-variate statistical analysis]] and hierarchical ascendant classification, or [[k-means clustering|''k''-means clustering]].{{cn|date=January 2023}}

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===Contrast transfer function===

Due to the nature of image formation in the electron microscope, [[Bright-field microscopy|bright-field]] TEM images are obtained using significant [[Focus (optics)|underfocus]]. This, along with features inherent in the microscope's lens system, creates blurring of the collected images visible as a [[point spread function]]. The combined effects of the imaging conditions are known as the [[contrast transfer function]] (CTF), and can be approximated mathematically as a function in reciprocal space. Specialized image processing techniques such as phase flipping and amplitude correction / [[Wiener filter|Wiener filtering]] can (at least partially)<ref name="Downing">{{Cite journal|vauthors=Downing KH, Glaeser RM |title=Restoration of weak phase-contrast images recorded with a high degree of defocus: the "twin image" problem associated with CTF correction |journal=Ultramicroscopy |volume=108 |issue=9 |pages=921–8 |date=August 2008 |pmid=18508199 |pmc=2694513 |doi=10.1016/j.ultramic.2008.03.004}}</ref>) correct for the CTF, and allow high resolution reconstructions.

===Three-dimensional reconstruction===

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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 [[wikibooks:Three_Dimensional_Electron_Microscopy/Initial_model#Random_Conical_Tilt|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|doi-access=free }}</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 [https://pubmed.ncbi.nlm.nih.gov/20888964/ orthogonal tilt reconstruction],<ref>{{cite journal |last1=Leschziner |first1=A |title=The orthogonal tilt reconstruction method. |journal=Methods in enzymology |date=2010 |volume=482 |pages=237-62 |doi=10.1016/S0076-6879(10)82010-5 |pmid=20888964}}</ref> +45 and −45 degrees. Pairs of particles corresponding to the same object at two different tilts (tilt pairs) are selected, and by following the parameters used in subsequent alignment and classification steps a three-dimensional reconstruction can be generated relatively easily. This is because the viewing angle (defined as three [[Euler angles]]) of each particle is known from the tilt geometry.

3D reconstructions from random conical tilt suffer from missing information resulting from a restricted range of orientations. Known as [the missing cone<ref>{{cite web |url=https://www.c-cina.org/research/algorithms/missing-cone/ the missing cone]}}</ref> (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 [[Negative stain|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<ref>{{cite journal |last1=Li |first1=Xueming |last2=Mooney |first2=Paul |last3=Zheng |first3=Shawn |last4=Booth |first4=Christopher R. |last5=Braunfeld |first5=Michael B. |last6=Gubbens |first6=Sander |last7=Agard |first7=David A. |last8=Cheng |first8=Yifan |title=Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM |journal=Nature Methods |date=June 2013 |volume=10 |issue=6 |pages=584–590 |doi=10.1038/nmeth.2472 |url=https://www.nature.com/articles/nmeth.2472 beam-induced|language=en movement]|issn=1548-7105}}</ref> makes collecting high-tilt images of samples in vitreous ice challenging.

===Map visualization and fitting===

Various software [[Software tools for molecular microscopy|programs]] are available that allow viewing the 3D maps. These often enable the user to manually dock in protein coordinates (structures from [[X-ray crystallography]] or NMR) of subunits into the electron density. Several programs can also fit subunits computationally.<ref>{{cite web |last1=Jamali |first1=Kiarash |last2=Käll |first2=Lukas |last3=Zhang |first3=Rui |last4=Brown |first4=Alan |last5=Kimanius |first5=Dari |last6=Scheres |first6=Sjors H.W. |title=Automated model building and protein identification in cryo-EM maps |date=16 May 2023 |doi=10.1101/2023.05.16.541002}}</ref>

===Single particle ICP-MS===

Single particle-induced coupled plasma-mass spectroscopy (SP-ICP-MS) is used in several areas where there is the possibility of detecting and quantifying suspended particles in samples of environmental fluids, assessing their migration, assessing the size of particles and their distribution, and also determining their stability in a given environment. SP-ICP-MS was designed for particle suspensions in 2000 by Claude Degueldre. He first tested this new methodology at the Forel Institute of the University of Geneva and presented this new analytical approach at the 'Colloid 2oo2' symposium during the spring 2002 meeting of the EMRS, and in the proceedings in 2003.<ref>C. Degueldre & P. -Y. Favarger, « Colloid analysis by single particle inductively coupled plasma-mass spectroscopy: a feasibility study », Colloids and Surfaces A: Physicochemical and Engineering Aspects, symposium C of the E-MRS 2002 Spring Meeting in Strasbourg, France, vol. 217, no 1, 28 avril 2003, p. 137–142 (ISSN 0927-7757, DOI 10.1016/S0927-7757(02)00568-X)</ref> This study presents the theory of SP ICP-MS and the results of tests carried out on clay particles (montmorillonite) as well as other suspensions of colloids. This method was then tested on thorium dioxide nanoparticles by Degueldre & Favarger (2004),<ref>C Degueldre et P. -Y Favarger, « Thorium colloid analysis by single particle inductively coupled plasma-mass spectrometry », Talanta, vol. 62, no 5, 19 avril 2004, p. 1051–1054 (ISSN 0039-9140, DOI 10.1016/j.talanta.2003.10.016</ref> zirconium dioxide by Degueldre et al (2004)<ref>C. Degueldre, P. -Y. Favarger et C. Bitea, « Zirconia colloid analysis by single particle inductively coupled plasma–mass spectrometry », Analytica Chimica Acta, vol. 518, no 1, 2 août 2004, p. 137–142 (ISSN 0003-2670, DOI 10.1016/j.aca.2004.04.015) </ref> and gold nanoparticles, which are used as a substrate in nanopharmacy, and published by Degueldre et al (2006).<ref>C. Degueldre, P. -Y. Favarger et S. Wold, « Gold colloid analysis by inductively coupled plasma-mass spectrometry in a single particle mode », Analytica Chimica Acta, vol. 555, no 2, 12 janvier 2006, p. 263–268 (ISSN 0003-2670, DOI 10.1016/j.aca.2005.09.021) </ref> Subsequently, the study of uranium dioxide nano- and micro-particles gave rise to a detailed publication, Ref. Degueldre et al (2006).<ref>C. Degueldre, P. -Y. Favarger, R. Rossé et S. Wold, « Uranium colloid analysis by single particle inductively coupled plasma-mass spectrometry », Talanta, vol. 68, no 3, 15 janvier 2006, p. 623–628 (ISSN 0039-9140, DOI 10.1016/j.talanta.2005.05.006, </ref> Since 2010 the interest for SP ICP-MS has exploded.

==Examples==