Automatic CTF correction for single particles based upon multivariate statistical analysis of individual power spectra

BackgroundThe transmission electron microscope is used to acquire structural information of macromolecular complexes. However, as any other imaging device, it introduces optical aberrations that must be corrected if high-resolution structural information is to be obtained. The set of all aberrations are usually modeled in Fourier space by the so-called Contrast Transfer Function (CTF). Before correcting for the CTF, we must first estimate it from the electron micrographs. This is usually done by estimating a number of parameters specifying a theoretical model of the CTF. This estimation is performed by minimizing some error measure between the theoretical Power Spectrum Density (PSD) and the experimentally observed PSD. The high noise present in the micrographs, the possible local minima of the error function for estimating the CTF parameters, and the cross-talking between CTF parameters may cause errors in the estimated CTF parameters.ResultsIn this paper, we explore the effect of these estimation errors on the theoretical CTF. For the CTF model proposed in [1] we show which are the most sensitive CTF parameters as well as the most sensitive background parameters. Moreover, we provide a methodology to reveal the internal structure of the CTF model (which parameters influence in which parameters) and to estimate the accuracy of each model parameter. Finally, we explore the effect of the variability in the detection of the CTF for CTF phase and amplitude correction.ConclusionWe show that the estimation errors for the CTF detection methodology proposed in [1] does not show a significant deterioration of the CTF correction capabilities of subsequent algorithms. All together, the methodology described in this paper constitutes a powerful tool for the quantitative analysis of CTF models that can be applied to other models different from the one analyzed here.

ACE: Automated CTF Estimation

Single particle tomography in EMAN2.

Contrast transfer function (CTF) estimation is essential to the data processing workflow of cryo-electron tomography (cryoET). Here, we present a protocol for CTF estimation of the cryoET tilt series with CTFMeasure. CTFMeasure can estimate the CTF parameters together with the absolute tilt angle offset of the sample. We describe steps for configuring the input files and estimating the CTF parameters and the absolute tilt angle offset of the input tilt series. We then detail procedures for visualizing the power spectra and analyzing the output files. For complete details on the use and execution of this protocol, please refer to Zhang etal.1.

This paper demonstrates a convenient experimental tech- nique for measuring the resolution of a digital imaging system. The spa- tial frequency response of the system is obtained by deriving the modu- lation transfer function (MTF) from the measured contrast transfer function (CTF). Basic signal-processing techniques are used to establish a relationship between the MTF and the CTF. Experimental methodology is discussed with respect to avoidance of shift variation in the CTF. Dis- crete Fourier transformation is applied for a power-spectrum analysis of the MTF. Using the CTF as a measure of performance, a series of ex- periments were carried out to characterize an imaging system based on an integrated CMOS camera. The experimental results showed that this approach can be applied in general, for the assessment of spatial reso- lution for digital imaging systems. © 1998 Society of Photo-Optical Instrumenta- tion Engineers. (S0091-3286(98)00609-6)

Application of template matching technique to particle detection in electron micrographs

Image formation in phase-contrast electron microscopy is governed by the contrast transfer function (CTF). The key parameter to tune the CTF is the defocus. A precise and unbiased estimate of the defocus is essential to determine the forward model and interpret high resolution images in cryo-TEM. We present an algorithm based on the weak-phase approximation for determination of the defocus and astigmatism of the objective lens from recorded images of an amorphous sample. The algorithm identifies Thon rings in the power spectrum density (PSD) and uses them to estimate astigmatism and defocus together with their uncertainties. For the astigmatism estimation we use a transformation to polar coordinates to fit ellipses to the Thon rings. Averaging the PSD over these ellipses reduces CTF estimation to a 1D problem and enhances the signal-to-noise ratio.

Electron tomography is a method for three-dimensional reconstruction of a specimen, based on its electron microscopical projections which are produced by tilting the specimen in the microscope about a fixed axis. After determining a common coordinate frame, a tomogram is computed by weighted backprojection. Higher resolution can be obtained by applying a contrast transfer function (CTF) correction to the projections. The tomographic data is further analyzed by extracting structural motifs from the raw tomograms, by selective alignment, multivariate statistical analysis, classification, and class-averaging, resulting in an increased signal-to-noise ratio and substantial data reduction. The volumetric data analysis facilitates the separation of a heterogeneous population of motifs into its constituents, and the visualization of the motifs in the raw tomograms by reassembly, replacing low signal-to-noise instances with higher signal-to-noise representations

The study was to describe and to compare the performance of 3D and 4D CBCT imaging modalities by measuring and analyzing the delivered dose and the image quality. The 3D (Chest) and 4D (Symmetry) CBCT Elekta XVI lung IGRT protocols were analyzed. Dose profiles were measured with TLDs inside a dedicated phantom. The dosimetric indicator cone‐beam dose index (CBDI) was evaluated. The image quality analysis was performed by assessing the contrast transfer function (CTF), the noise power spectrum (NPS) and the noise‐equivalent quanta (NEQ). Artifacts were also evaluated by simulating irregular breathing variations. The two imaging modalities showed different dose distributions within the phantom. At the center, the 3D CBCT delivered twice the dose of the 4D CBCT. The CTF was strongly reduced by motion compared to static conditions, resulting in a CTF reduction of 85% for the 3D CBCT and 65% for the 4D CBCT. The amplitude of the NPS was two times higher for the 4D CBCT than for the 3D CBCT. In the presence of motion, the NEQ of the 4D CBCT was 50% higher than the 3D CBCT. In the presence of breathing irregularities, the 4D CBCT protocol was mainly affected by view‐aliasing artifacts, which were typically cone‐beam artifacts, while the 3D CBCT protocol was mainly affected by duplication artifacts. The results showed that the 4D CBCT ensures a reasonable dose and better image quality when moving targets are involved compared to 3D CBCT. Therefore, 4D CBCT is a reliable imaging modality for lung free‐breathing radiation therapy.PACS number(s): 87.57.C‐, 87.57.uq, 87.53.Ly

Power spectrum analysis is one of the important part of digital signal processing, various characteristics of the main signal in the frequency domain, is designed based on the limited data and extract the useful signal is submerged in the noise in the frequency domain. In this paper, the VB language to realize the power spectrum analysis can extract the useful signal was drowned in noise in frequency domain based on power spectrum, frequency structure and physical meaning analysis reflected the more obvious, monitoring and diagnosis in rotating machinery fault, power spectrum analysis is very wide.

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

FindEM—a fast, efficient program for automatic selection of particles from electron micrographs

Multivariate statistical analysis of three-dimensional cross-bridge motifs in insect flight muscle

Recent advances in photon counting detection technology have led to significant research interest in X-ray imaging. As a tutorial level review, this paper covers a wide range of aspects related to X-ray photon counting detector characterization. The tutorial begins with a detailed description of the working principle and operating modes of a pixelated X-ray photon counting detector with basic architecture and detection mechanism. Currently available methods and techniques for charactering major aspects including energy response, noise floor, energy resolution, count rate performance (detector efficiency), and charge sharing effect of photon counting detectors are comprehensively reviewed. Other characterization aspects such as point spread function (PSF), line spread function (LSF), contrast transfer function (CTF), modulation transfer function (MTF), noise power spectrum (NPS), detective quantum efficiency (DQE), bias voltage, radiation damage, and polarization effect are also remarked. A cadmium telluride (CdTe) pixelated photon counting detector is employed for part of the characterization demonstration and the results are presented. This review can serve as a tutorial for X-ray imaging researchers and investigators to understand, operate, characterize, and optimize photon counting detectors for a variety of applications.

2.02 - Physical Parameters of Image Quality