ASC-G4, an algorithm to calculate advanced structural characteristics of G-quadruplexes.

ASC-G4 is an algorithm for the calculation of the advanced structural characteristics of G-quadruplexes (G4). It allows the unambiguous determination of the intramolecular G4 topology, based on the oriented strand numbering. It also resolves the ambiguity in the determination of the guanine glycosidic configuration. With this algorithm, we showed that the use of the C3' or C5' atoms to calculate the groove width in G4 is more appropriate than the P atoms and that the groove width does not always reflect the space available within the groove. For the latter, the minimum groove width is more appropriate. The application of ASC-G4 to 207 G4 structures guided the choices made for the calculations. A website based on ASC-G4 (http://tiny.cc/ASC-G4) was created, where the user uploads his G4 structure and gets its topology, the types of its loops and their lengths, the presence of snapbacks and bulges, the distribution of guanines in the tetrads and strands, the glycosidic configuration of these guanines, their rise, the groove widths, the minimum groove widths, the tilt and twist angles, the backbone dihedral angles, etc. It also provides a large number of atom-atom and atom-plane distances that are relevant to evaluating the quality of the structure.

MMX-I: data-processing software for multimodal X-ray imaging and tomography.

A new multi-platform freeware has been developed for the processing and reconstruction of scanning multi-technique X-ray imaging and tomography datasets. The software platform aims to treat different scanning imaging techniques: X-ray fluorescence, phase, absorption and dark field and any of their combinations, thus providing an easy-to-use data processing tool for the X-ray imaging user community. A dedicated data input stream copes with the input and management of large datasets (several hundred GB) collected during a typical multi-technique fast scan at the Nanoscopium beamline and even on a standard PC. To the authors' knowledge, this is the first software tool that aims at treating all of the modalities of scanning multi-technique imaging and tomography experiments.

Wet-STEM tomography: principles, potentialities and limitations.

The characterization of biological and inorganic materials by determining their three-dimensional structure in conditions closer to their native state is a major challenge of technological research. Environmental scanning electron microscopy (ESEM) provides access to the observation of hydrated samples in water environments. Here, we present a specific device for ESEM in the scanning transmission electron microscopy mode, allowing the acquisition of tilt-series suitable for tomographic reconstructions. The resolution which can be obtained with this device is first determined. Then, we demonstrate the feasibility of tomography on wet materials. The example studied here is hydrophilic mesoporous silica (MCM-41). Finally, the minimum thickness of water which can be detected is calculated from Monte Carlo simulations and compared with the resolution expected in the tomograms.

Three-dimensional chemical mapping by EFTEM-TomoJ including improvement of SNR by PCA and ART reconstruction of volume by noise suppression.

Electron tomography is becoming one of the most used methods for structural analysis at nanometric scale in biological and materials sciences. Combined with chemical mapping, it provides qualitative and semiquantitative information on the distribution of chemical elements on a given sample. Due to the current difficulties in obtaining three-dimensional (3D) maps by energy-filtered transmission electron microscopy (EFTEM), the use of 3D chemical mapping has not been widely adopted by the electron microscopy community. The lack of specialized software further complicates the issue, especially in the case of data with a low signal-to-noise ratio (SNR). Moreover, data interpretation is rendered difficult by the absence of efficient segmentation tools. Thus, specialized software for the computation of 3D maps by EFTEM needs to include optimized methods for image series alignment, algorithms to improve SNR, different background subtraction models, and methods to facilitate map segmentation. Here we present a software package (EFTEM-TomoJ, which can be downloaded from http://u759.curie.fr/fr/download/softwares/EFTEM-TomoJ), specifically dedicated to computation of EFTEM 3D chemical maps including noise filtering by image reconstitution based on multivariate statistical analysis. We also present an algorithm named BgART (for background removing algebraic reconstruction technique) allowing the discrimination between background and signal and improving the reconstructed volume in an iterative way.

Protocol for automated multivariate quantitative-image-based cytometry analysis by fluorescence microscopy of asynchronous adherent cells.

Here, we present a protocol for multivariate quantitative-image-based cytometry (QIBC) analysis by fluorescence microscopy of asynchronous adherent cells. We describe steps for the preparation, treatment, and fixation of cells, sample staining, and imaging for QIBC. We then detail image analysis with our open source Fiji script developed for QIBC and present multiparametric data visualization. Our QIBC Fiji script integrates modern artificial-intelligence-based tools, applying deep learning, for robust automated nuclei segmentation with minimal user adjustments, a major asset for efficient QIBC analysis. For complete details on the use and execution of this protocol, please refer to Besse et al. (2023).1.

SEM3De: image restoration for FIB-SEM.

Motivation: FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. Results: With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. Availability and implementation: SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

3D analysis of the morphology and spatial distribution of nitrogen in nitrogen-doped carbon nanotubes by energy-filtered transmission electron microscopy tomography.

We present here the application of the energy-filtered transmission electron microscopy (EFTEM) in the tomographic mode to determine the precise 3D distribution of nitrogen within nitrogen-doped carbon nanotubes (N-CNTs). Several tilt series of energy-filtered images were acquired on the K ionization edges of carbon and nitrogen on a multiwalled N-CNT containing a high amount of nitrogen. Two tilt series of carbon and nitrogen 2D maps were then calculated from the corresponding energy-filtered images by using a proper extraction procedure of the chemical signals. Applying iterative reconstruction algorithms provided two spatially correlated C and N elemental-selective volumes, which were then simultaneously analyzed with the shape-sensitive reconstruction deduced from Zero-Loss recordings. With respect to the previous findings, crucial information obtained by analyzing the 3D chemical maps was that, among the two different kind of arches formed in these nanotubes (transversal or rounded ones depending on their morphology), the transversal arches contain more nitrogen than do the round ones. In addition, a detailed analysis of the shape-sensitive volume allowed the observation of an unexpected change in morphology along the tube axis: close to the round arches (with less N), the tube is roughly cylindrical, whereas near the transversal ones (with more N), its shape changes to a prism. This relatively new technique is very powerful in the material science because it combines the ability of the classical electron tomography to solve 3D structures and the chemical selectivity of the EFTEM imaging.

MITF activity is regulated by a direct interaction with RAF proteins in melanoma cells.

The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven mouse melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF partner. Interestingly, this interaction is conserved among the RAF protein kinase family since BRAF/MITF and CRAF/MITF complexes were also observed in the cytosol of NRAS-mutated mouse melanoma cells. The interaction occurs through the kinase domain of RAF proteins. Importantly, endogenous BRAF/MITF complexes were also detected in BRAF-mutated human melanoma cells. RAF/MITF complexes modulate MITF nuclear localization by inducing an accumulation of MITF in the cytoplasm, thus negatively controlling its transcriptional activity. Taken together, our study highlights a new level of regulation between two major mediators of melanoma progression, MITF and the MAPK/ERK pathway, which appears more complex than previously anticipated.

Determination of Microtubule Lattice Parameters from Cryo-electron Microscope Images Using TubuleJ.

The α-ß tubulin heterodimer undergoes subtle conformational changes during microtubule assembly. These can be modulated by external factors, whose effects on microtubule structure can be characterized on 2D views obtained by cryo-electron microscopy. Analysis of microtubule images is facilitated if they are straight enough to interpret and filter their image Fourier transform, which provide useful information concerning the arrangement of tubulin molecules inside the microtubule lattice. Here, we describe the use of the TubuleJ software to straighten microtubules and determine their lattice parameters. Basic 3D reconstructions can be performed to evaluate the relevance of these parameters. This approach can be used to analyze the effects of nucleotide analogues, drugs or MAPs on microtubule structure, or to select microtubule images prior to high-resolution 3D reconstructions.

Marker-free image registration of electron tomography tilt-series.

BACKGROUND: Tilt series are commonly used in electron tomography as a means of collecting three-dimensional information from two-dimensional projections. A common problem encountered is the projection alignment prior to 3D reconstruction. Current alignment techniques usually employ gold particles or image derived markers to correctly align the images. When these markers are not present, correlation between adjacent views is used to align them. However, sequential pairwise correlation is prone to bias and the resulting alignment is not always optimal. RESULTS: In this paper we introduce an algorithm to find regions of the tilt series which can be tracked within a subseries of the tilt series. These regions act as landmarks allowing the determination of the alignment parameters. We show our results with synthetic data as well as experimental cryo electron tomography. CONCLUSION: Our algorithm is able to correctly align a single-tilt tomographic series without the help of fiducial markers thanks to the detection of thousands of small image patches that can be tracked over a short number of images in the series.

Iterative Bayesian denoising based on variance stabilization using Contourlet Transform with Sharp Frequency Localization: application to EFTEM images.

BACKGROUND: Due to the presence of high noise level in tomographic series of energy filtered transmission electron microscopy (EFTEM) images, alignment and 3D reconstruction steps become so difficult. To improve the alignment process which will in turn allow a more accurate and better three dimensional tomography reconstructions, a preprocessing step should be applied to the EFTEM data series. RESULTS: Experiments with real EFTEM data series at low SNR, show the feasibility and the accuracy of the proposed denoising approach being competitive with the best existing methods for Poisson image denoising. The effectiveness of the proposed denoising approach is thanks to the use of a nonparametric Bayesian estimation in the Contourlet Transform with Sharp Frequency Localization Domain (CTSD) and variance stabilizing transformation (VST). Furthermore, the optimal inverse Anscome transformation to obtain the final estimate of the denoised images, has allowed an accurate tomography reconstruction. CONCLUSION: The proposed approach provides qualitative information on the 3D distribution of individual chemical elements on the considered sample.

Compressed sensing for STEM tomography.

A central challenge in scanning transmission electron microscopy (STEM) is to reduce the electron radiation dosage required for accurate imaging of 3D biological nano-structures. Methods that permit tomographic reconstruction from a reduced number of STEM acquisitions without introducing significant degradation in the final volume are thus of particular importance. In random-beam STEM (RB-STEM), the projection measurements are acquired by randomly scanning a subset of pixels at every tilt view. In this work, we present a tailored RB-STEM acquisition-reconstruction framework that fully exploits the compressed sensing principles. We first demonstrate that RB-STEM acquisition fulfills the "incoherence" condition when the image is expressed in terms of wavelets. We then propose a regularized tomographic reconstruction framework to recover volumes from RB-STEM measurements. We demonstrate through simulations on synthetic and real projection measurements that the proposed framework reconstructs high-quality volumes from strongly downsampled RB-STEM data and outperforms existing techniques at doing so. This application of compressed sensing principles to STEM paves the way for a practical implementation of RB-STEM and opens new perspectives for high-quality reconstructions in STEM tomography.

Overview of chemical imaging methods to address biological questions.

Chemical imaging offers extensive possibilities for better understanding of biological systems by allowing the identification of chemical components at the tissue, cellular, and subcellular levels. In this review, we introduce modern methods for chemical imaging that can be applied to biological samples. This work is mainly addressed to the biological sciences community and includes the bases of different technologies, some examples of its application, as well as an introduction to approaches on combining multimodal data.

Scanning transmission electron microscopy through-focal tilt-series on biological specimens.

Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.

Electron microscope tomography of native membranes.

Membrane proteins are often present in low amounts in cells. Their function can be modulated by interactions with other proteins. Moreover, these complexes can be transiently formed, thus making them difficult to be isolated and to be purified. One way to overcome these difficulties is to visualize these complexes in situ in the cells. For such purpose, electron microscopy coupled to tomography is a promising approach that has been developed over the last decades.Mitochondria are a good example of organelles where many membrane proteins form different functional complexes within the outer and the inner membranes. The latter is either close to the former or projects within the matrix to form cristae. Structure of these cristae involves different proteins and can vary from lamellar to tubular forms in normal mitochondria. In pathological conditions, other mitochondrial morphologies have been described, for instance, vesicular structures for inner boundary membrane have been observed.

Electron tomography study of isolated human centrioles.

Centrioles are components of the centrosome, which is present in most eukaryotic cells (from protozoa to mammals). They organize the microtubule skeleton during interphase and the mitotic spindle during cell division. In ciliate cells, centrioles form basal bodies that are involved in cellular motility. Despite their important roles in biology, the detailed structure of centrioles remains obscure. This work contributes to a more complete model of centriole structure. The authors used electron tomography of isolated centrosomes from the human lymphoblast KE37 to explore the details of subdistal appendages and centriole lumen organization in mother centrioles. Their results reveal that each of the nine subdistal appendages is composed of two halves (20 nm diameter each) fused in a 40 nm tip that extends 100 nm from where it anchors to microtubules. The centriole lumen is filled at the distal domain by a 45 nm periodic stack of rings. Each ring has a 30 nm diameter, is 15 nm thick, and appears to be tilted at 53 degrees perpendicular to the centriole axis. The rings are anchored to microtubules by arms. Based on their results, the authors propose a model of the mother centriole distal structure.

Multiple-axis tomography: applications to basal bodies from Paramecium tetraurelia.

BACKGROUND INFORMATION: Transmission electron tomography is becoming a powerful tool for studying subcellular components of cells. Classical approaches for electron tomography consist of recording images along a single-tilt axis. This approach is being improved by dual-axis reconstructions and/or high-tilt devices (tilt angle>+/-60 degrees) on microscopes to compensate part of the information loss due to the 'missing wedge' phenomena. RESULTS: In the present work we have evaluated the extension of the dual-axis technique to a multiple-axis approach, and we demonstrate a freely available plug-in for the Java-based freeware image-analysis software ImageJ. Our results from phantom and experimental data sets from Paramecium tetraurelia epon-embedded sections have shown that multiple-axis tomography achieves results equivalent to those obtained by dual-axis approach without the requirement for high-tilt devices. CONCLUSIONS: This new approach allows performance of high-resolution tomography, avoiding the need for high-tilt devices, and therefore will increase the access of electron tomography to a larger community.

Smart 3D-FISH: automation of distance analysis in nuclei of interphase cells by image processing.

BACKGROUND: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved three-dimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function. METHODS: 3D-FISH protocol, confocal microscopy, and digital image analysis were used. RESULTS: New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4',6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of spot loci in the nucleus and inter-spot distance. Results from a pilot experiment using Smart 3D-FISH on ENL, MLL, and AF4 genes in two lymphoblastic cell lines were satisfactory and consistent with data published in the literature. CONCLUSION: Smart 3D-FISH should greatly facilitate image processing and analysis of 3D-FISH images by providing a useful tool to overcome the laborious task of image segmentation based on user-defined parameters and decrease subjectivity in data analysis. It is available as a set of plugins for ImageJ software.

Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage.

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.