Cédric Messaoudi

Marker-free image registration of electron tomography tilt-series

BackgroundTilt 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.ResultsIn 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.ConclusionOur 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.

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

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.

Electron tomography of biological samples

Electron tomography allows computing three-dimensional (3D) reconstructions of objects from their projections recorded at several angles. Combined with transmission electron microscopy, electron tomography has contributed greatly to the understanding of subcellular structures and organelles. Performed on frozen-hydrated samples, electron tomography has yielded useful information about complex biological structures. Combined with energy filtered transmission electron microscopy (EFTEM) it can be used to analyze the spatial distribution of chemical elements in biological or material sciences samples. In the present review, we present an overview of the requirements, applications, and perspectives of electron tomography in structural biology.

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° 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. Microsc. Res. Tech., 2009.

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°) on microscopes to compensate part of the information loss due to the ‘missing wedge’ phenomena.

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.

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 (Adrian et al., 1998), which reduces the radiation damage while preserving a high signal‐to‐noise ratio (De Carlo et al., 2002). 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.

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

The MMX-I open-source software has been developed for processing and reconstruction of large multimodal X-ray imaging and tomography datasets. The recent version of MMX-I is optimized for scanning X-ray fluorescence, phase-, absorption- and dark-field contrast techniques. This, together with its implementation in Java, makes MMX-I a versatile and friendly user tool for X-ray imaging.

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.

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.