Nuclear architecture and the induction of chromosomal aberrations

Progress in fluorescence in situ hybridization, three dimensional microscopy and image analysis has provided the means to study the three-dimensional structure and distribution of chromosome territories within the cell nucleus. In this contribution, we summarize the present state of knowledge of the territorial organization of interphase chromosomes and their topological relationships with other macromolecular domains in the human cell nucleus, and present data from computer simulations of chromosome territory distributions. On this basis, we discuss models of chromosome territory and nuclear architecture and topological consequences for the formation of chromosome exchanges.     

Nonrigid registration of 3-d multichannel microscopy images of cell nuclei.

We present an intensity-based nonrigid registration approach for the normalization of 3-D multichannel microscopy images of cell nuclei. A main problem with cell nuclei images is that the intensity structure of different nuclei differs very much; thus, an intensity-based registration scheme cannot be used directly. Instead, we first perform a segmentation of the images from the cell nucleus channel, smooth the resulting images by a Gaussian filter, and then apply an intensity-based registration algorithm. The obtained transformation is applied to the images from the nucleus channel as well as to the images from the other channels. To improve the convergence rate of the algorithm, we propose an adaptive step length optimization scheme and also employ a multiresolution scheme. Our approach has been successfully applied using 2-D cell-like synthetic images, 3-D phantom images as well as 3-D multichannel microscopy images representing different chromosome territories and gene regions. We also describe an extension of our approach, which is applied for the registration of 3D + t (4-D) image series of moving cell nuclei.     

Effect of CMAS Deposits on MOCVD Coatings in the System Y2O3–ZrO2: Phase Relationships

Three metal‐organic chemical vapor deposition (MOCVD) coatings (ZrO₂, Y₂O₃·ZrO₂, Y₂O₃) were studied in contact with model CMAS to investigate the microstructural evolution and phase formation. The MOCVD coatings were covered with CMAS powder deposits and annealed for 1 h at 1250°C, respectively. The ZrO₂ coating was completely infiltrated by CMAS, whereas the yttria containing coatings show a higher resistance against CMAS infiltration. This is explained by the formation of a continuous oxyapatite layer in the reaction zone of the coating and the CMAS deposit. The Y₂O₃·ZrO₂ coating shows the best infiltration resistance despite the fact that the Y₂O₃–CMAS sample is the only completely crystallized. As crystallization products, oxyapatite, melilite, anorthite as well as new garnets bearing all available cations were formed. The garnet phase was confirmed by XRD and TEM. EDS measurements were used to calculate structural formula A₃B₂T₃O₁₂ (A = Ca, Y, Zr; B = Mg, Al; T = Al, Si).     

High-Temperature Hydroxylation and Surface Corrosion of 2/1-Mullite Single Crystals in Water Vapor Environments

2/1-mullite single crystal (001) plates with thicknesses between 0.9 and 1.9 mm were exposed for 1.5, 3, 6, and 12 h at 1670°C to a slowly flowing (100 mL/min) water-rich gas mixture (O₂/H₂O 80/20). Under the given experimental conditions, 2/1-mullite yielded significant amounts of structurally bound OH groups across the bulk and decomposition of the crystal surface on a micrometer scale. Decomposition products are (i) sodium-containing silicon-rich alumino silicate glass formed from melt and (ii) α-alumina, which crystallizes within melt cavities. The crystal plates that are free of any OH absorption before the corrosion experiments show a steep increase in OH absorption intensity up to 3 h of corrosion and a flattening toward longer times of exposure. The evaluation of OH intensity profiles implies an effective diffusion coefficient D _H in the range between 1.5 and 2.5 × 10⁻⁷ cm²/s.     

Identifying virus-cell fusion in two-channel fluorescence microscopy image sequences based on a layered probabilistic approach

The entry process of virus particles into cells is decisive for infection. In this work, we investigate fusion of virus particles with the cell membrane via time-lapse fluorescence microscopy. To automatically identify fusion for single particles based on their intensity over time, we have developed a layered probabilistic approach. The approach decomposes the action of a single particle into three abstractions: the intensity over time, the underlying temporal intensity model, as well as a high level behavior. Each abstraction corresponds to a layer and these layers are represented via stochastic hybrid systems and hidden Markov models. We use a maxbelief strategy to efficiently combine both representations. To compute estimates for the abstractions we use a hybrid particle filter and the Viterbi algorithm. Based on synthetic image sequences, we characterize the performance of the approach as a function of the image noise. We also characterize the performance as a function of the tracking error. We have also successfully applied the approach to real image sequences displaying pseudotyped HIV-1 particles in contact with host cells and compared the experimental results with ground truth obtained by manual analysis.     

Contactless determination of nuclear compressibility using 3D image- and model-based analysis of drug-induced cellular deformation

Mechanical properties of the chromatin-bearing nucleus in normal and pathological cells are of general interest for epigenetics and medicine. Conventional techniques for quantitative measurements of material properties of cellular matter are based on application of controlled forces onto the cellular or nuclear boundary and do not allow probing intracellular structures that are not directly accessible for physical contact inside the living cell. In this work, we present a novel approach for contactless determination of the nuclear compressibility (i.e. the Poisson's ratio ν) in living cells by means of image- and model-based analysis of drug-induced cell deformation. The Poisson's ratio of the HeLa cell nucleus is determined from time-series of 3D images as a parameter of constitutive model that minimizes the dissimilarity between the numerically predicted and experimentally observed images.     

A database system for comparative genomic hybridization analysis

Comparative genomic hybridization (CGH) is a molecular cytogenetic analysis method that allows the detection of chromosomal imbalances in entire genomes. The CGH approach is used in cancer research to identify over- and under-representations of chromosomal regions. To search for and analyze tumor-relevant aberration patterns in CGH data, we designed, implemented, and deployed a relational database system. This project is part of a more complex and comprehensive effort to compile, integrate, fuse, and analyze biological and clinical data from heterogeneous and distributed information sources. In this article, we discuss the obstacles and pitfalls that were encountered in the design process, describe the resulting CGH database model and the underlying technical infrastructure, and present the first results based on mining the CGH database     

High-precision distance measurements and volume-conserving segmentation of objects near and below the resolution limit in three-dimensional confocal fluorescence microscopy

This study presents a method for high-precision distance measurements and for the volume-conserving segmentation of fluorescent objects with a size of the order of the microscopic observation volume. The segmentation was performed via a model-based approach, using an algorithm that was calibrated by the microscopic point spread function. Its performance was evaluated for three different fluorochromes using model images and fluorescent microspheres as test targets. The fundamental limits which the microscopic imaging process imposes on the accuracy of volume and distance measurements were evaluated in detail. A method for the calibration of the axial stepwidth of a confocal microscope is presented. The results suggest that in biological applications, 3D distances and radii of objects in cell nuclei can be determined with an accuracy of ≤ 60 nm. Using objects of different spectral signature, 3D distance measurements substantially below the lateral half width of the confocal point spread function are feasible. This is shown both theoretically and experimentally.     

Spatial distributions of early and late replicating chromatin in interphase chromosome territories

The surface area of chromosome territories has been suggested as a preferred site for genes, specific RNAs, and accumulations of splicing factors. Here, we investigated the localization of sites of replication within individual chromosome territories. In vivo replication labeling with thymidine analogues IdUrd and CldUrd was combined with chromosome painting by fluorescent in situ hybridization on three-dimensionally preserved human fibroblast nuclei. Spatial distributions of replication labels over the chromosome territory, as well as the territory volume and shape, were determined by 3D image analysis. During late S-phase a previously observed shape difference between the active and inactive X-chromosome in female cells was maintained, while the volumes of the two territories did not differ significantly. Domains containing early or mid to late replicating chromatin were distributed throughout territories of chromome 8 and the active X. In the inactive X-chromosome early replicating chromatin was observed preferentially near the territory surface. Most important, we established that the process of replication takes place in foci throughout the entire chromosome territory volume, in early as well as in late S-phase. This demonstrates that activity of macromolecular enzyme complexes takes place throughout chromosome territories and is not confined to the territory surface as suggested previously.     

An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH)

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromosomes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.